Introduction
Plants and herbivore insects have undergone co-evolution for more than 350 million years (War et al., Reference War, Paulraj, Ahmad, Buhroo, Hussain, Ignacimuthu and Sharma2012), and it is widely known that biotic stress caused by insect infestation can affect the growth and development of host plants. As one of the key indicators of plant growth, photosynthesis is of vital importance. In most cases, insect infestation is associated with a reduction in photosynthesis. For example, the previous study explored the relationships between biotic damage and the expression of photosynthesis-related genes in eight plant species, which strongly supported the universal down-regulation of these genes and indicated that their expression was an adaptive response to biotic stress (Bilgin et al., Reference Bilgin, Zavala, Zhu, Clough, Ort and Delucia2010). Also, the bug feeding led to a rapid and substantial decrease in photosynthesis in both Brassica oleracea L. var. botrytis and Phaseolus vulgaris L. leaves, and further in-depth studies demonstrated that Murgantia histrionica (Hahn) oviposition had the same effect (Velikova et al., Reference Velikova, Salerno, Frati, Peri, Conti, Colazza and Loreto2010). A similar investigation had been conducted in Scots pine, showing that the photosynthetic rate of oviposition-induced plants was remarkably lower than that of the control (Schröder et al., Reference Schröder, Forstreuter and Hilker2005).
Conversely, to counter insect attacks, several defense responses mainly categorized as biochemical, molecular and behavioral aspects are induced after the stimulation of stress factors (Boller and Felix, Reference Boller and Felix2009). In biochemical and molecular aspects, step-wise reactions generate (Erb et al., Reference Erb, Meldau and Howe2012). First, plants perceive herbivore insect attack by recognizing alien infectious signals via conserved patterns of microbial molecules. After an attacker is recognized, different signaling cascades are activated and reprogrammed, mainly involving the transduction of cellular signals to downstream signaling molecules, and then the defense responses culminate. During this process, plants produce various defensive compounds necessary for their growth under the attack of herbivore insects (Schafer et al., Reference Schafer, Grassian, Song, Jiang, Gerhart-Hines, Irie, Gao, Puigserver and Brugge2009). Among these defensive compounds, secondary metabolites, especially enzymatic antioxidants are of great concern (War et al., Reference War, Buhroo, Hussain, Ahmad, Nair, Sharma, Mérillon and Ramawat2020). There are numerous examples of antioxidants participating in defenses against insect herbivores, particularly, classical antioxidants such as phenols (Bi et al., Reference Bi, Felton, Murphy, Howles, Dixon and Lamb1997), reactive oxygen species (ROS) (Kawano, Reference Kawano2003), proline (Qamar et al., Reference Qamar, Mysore and Senthil-Kumar2015), hydrogen peroxide (H2O2) (Levine et al., Reference Levine, Tenhaken, Dixon and Lamb1994), malondialdehyde (Zhang et al., Reference Zhang, Hua and Zhang2008) and so on. Many solid foundations have been laid that these non-enzymatic antioxidants were dramatically increased after insect herbivory (War et al., Reference War, Paulraj, Ahmad, Buhroo, Hussain, Ignacimuthu and Sharma2012; Golan et al., Reference Golan, Rubinowska and Górska-Drabik2013; Akram et al., Reference Akram, Shafiq and Ashraf2017).
One of the important defense mechanisms of plants against herbivorous insect infestation is the activation of antioxidative enzymes. Among which, peroxidase, superoxide dismutase (SOD) and catalase play important roles (Rani and Jyothsna, Reference Rani and Jyothsna2010). SOD acts in removing superoxide anion-free radicals (Sudhakar et al., Reference Sudhakar, Lakshmi and Giridarakumar2001). Surveys such as that conducted by Yang et al. (Reference Yang, Han, Li, Li, Ali and Hou2017) have shown that the activity of SOD increased in response to Nilaparvata lugens (Stål) infestation (Yang et al., Reference Yang, Han, Li, Li, Ali and Hou2017). In contrast, peroxidases are monomeric hemoproteins within cells and commonly existed in plants. A numbers of processes acting in direct or indirect role in plant defense are regulated by peroxidases (War et al., Reference War, Paulraj, Ahmad, Buhroo, Hussain, Ignacimuthu and Sharma2012). For example, transgenic tobacco, tomato and tree species with tobacco anionic peroxidase overexpression conferred resistance to several insects (Dowd and Lagrimini, Reference Dowd and Lagrimini1997). Also, it has been previously observed that Spodoptera frugiperda (JE Smith) and Blissus occiduus (Barber) infestation resulted in elevated peroxidase activity in corns and buffalo grass, respectively (Heng-Moss et al., Reference Heng-Moss, Sarath, Baxendale, Novak, Bose, Ni and Quisenberry2004; Chen et al., Reference Chen, Ni and Buntin2009). In addition to peroxidase and SOD, catalase also functions in plant defense. In catalase-deficient Arabidopsis thaliana (Heynh), the expression of defense-related genes drastically decreased (Vandenabeele et al., Reference Vandenabeele, Vanderauwera, Vuylsteke, Rombauts, Langebartels, Seidlitz, Zabeau, Van Montagu, Inzé and Van Breusegem2004). Besides, the fluctuation of total soluble protein and sugar important for plant growth and maintenance also affects its defense response against insect herbivory, and much evidence showed that the increase in protein and sugar contents mediated plant resistance to herbivore infestation (de Souza Cândido et al., Reference de Souza Cândido, Pinto, Pelegrini, Lima, Silva, Pogue, Grossi-de-Sá and Franco2011; Bolouri Moghaddam and Van den Ende, Reference Bolouri Moghaddam and Van den Ende2012).
As one of the most important functional and dietetic fruit crops in arid and semi-arid regions outspreading from the Arabian Peninsula to North Africa and the Middle East, the date palm Phoenix dactylifera L. has been cultivated for thousands of years (Memon et al., Reference Memon, Noonari, Kalwar and Sial2015), and its production dramatically increased more than 100-fold (Chao and Krueger, Reference Chao and Krueger2007). Among all date palm producing countries, Pakistan ranked seventh, indicating its economic importance (Nadeem et al., Reference Nadeem, Qureshi, Ugulu, Riaz, Ain, Khan, Ahmad, Ashfaq, Bashir and Dogan2019). Being rich in nutrition, date palms provide a perfect choice for insect feeding (Haider et al., Reference Haider, Khan, Jaskani, Naqvi, Mateen, Shahzad and Abbas2018). Among the pests infecting date palms, Rhynchophorus ferrugineus (Oliver) (red palm weevil (RPW)) is the most common one. It belongs to the Curculionidae of Coleoptera, and is an invasive forest pest that seriously endangers various plants. It was reported that the RPW originated from India and broken out in many places, including North Africa, Middle East and Southern Europe during the past few decades (Chao and Krueger, Reference Chao and Krueger2007). The RPW was identified as the first category serious pest by the Food and Agriculture Organization and caused severe injury to the date palms. Surveys conducted by Mohamed et al. (Reference Mohamed, Pathak, Farooq and White2019) showed that the infestation of RPW inside the trunk of date palms led to its collapse within few months (Mohamed et al., Reference Mohamed, Pathak, Farooq and White2019). Moreover, the RPW invasion caused an annual loss of $1.74–8.69 million in the East of the Kingdom of Saudi Arabia (El-Sabea et al., Reference El-Sabea, Faleiro and Abo-El-Saad2009). In Pakistan, RPW was identified and characterized in four provinces, Punjab, Sindh, Khyber Pakhtunkhwa and Baluchistan, also posing a huge threat to agriculture (Mujahid et al., Reference Mujahid, Ahmad, Arif, Nazir and Giblin-Davis2018).
Several lines of evidence have suggested that the RPW prefers to feed on date palms. No such survey has been conducted to investigate the preference of RPW to different cultivars. The host plants exhibit a diversity of defense responses upon RPW infestations, including changes in antioxidants and anti-oxidase activities. Hereby, the present study first aims at comparing the feeding preference of RPW to 11 date palm cultivars, and then analyzes the biochemical responses of date palms under RPW infestation. It is expected that this study enhances our understanding of the biochemical mechanism of date palms under RPW attack, and further contributes to its control.
Materials and methods
Feeding preference analysis
Fifty grams fresh leaves of 11 commercial date palm cultivars, Hillawi, Mozawati, Kechanr, Aseel, Khudravi, Shamrani, Dhaki, Zaidi, Zeri, Kobra and Denda, were collected from grown orchards and used for olfactometer assay. The feeding preference experiment was conducted using the Inlet Odour Source System. Briefly, fresh date palm pieces of cultivars and the control (Kobra and Denda cultivars) were kept on each side of the two-arm olfactometer. The test RPW was collected from a previous indoor-maintained population and 40 gravid female adults of 15 days old were used for the behavioral analysis. After 10 min, the insects trapped in each side of an olfactometer were collected and counted. The attraction rate of cultivar ‘X’ (%) was calculated as ‘(the number of RPW in cultivar X − ram/the total number of RPW in two-arms) × 100’. We counted the number of laying eggs of RPW on each cultivar and calculated the hatchability (%) as follows ‘(the hatched eggs/total laying eggs) × 100’. Each measurement was repeated three times.
Fecundity and egg hatchability
The new leaves of 11 cultivars were collected and separately kept in 11 plastic cages with a dimension of 35 cm × 40 cm × 60 cm, respectively. Three leaves were kept in pots of water to keep humid per cage. Then, ten gravid females and two male weevils were released into these cages. After 1 week, the adults were removed. To establish number of eggs and hatchability, the eggs laid by RPW females and hatched larvae on each cultivar were counted, respectively. We calculated the hatchability (%) as follows ‘(the hatched larvae/total laid eggs) × 100’. Each measurement was repeated three times.
Sample preparation
RPW were collected from the Punjab province of Pakistan and reared in an artificial climate incubator. Briefly, 15 RPW first-instar larvae within 24 h hatching were fed on the fresh leaves of date palm cultivar Hillawi, and the healthy date palms of the same age without RPW infestation were used as control. After 30–35 days, the infested and uninfested leaves of date palms were collected and chopped into small pieces for subsequent biochemical analysis, respectively.
Determination of biochemical substance contents
Total soluble protein
Total soluble protein of date palm leaves was extracted from 0.5 g leaves. Briefly, a fresh sample was ground in 5 ml of 50 mM cooled phosphate buffer (pH 7.8), and the homogenate was centrifuged at 15,000 rpm for 15 min. The supernatant was used for soluble protein determination. We measured the soluble proteins using the Bradford method (Bradford, Reference Bradford1976). A total of 100 μl supernatant was transferred into a microfuge tube and mixed with 1 ml of Bradford reagent. The sample was incubated at 37°C for 10–15 min followed by absorbance measurements at 595 nm using the Picodrop spectrophotometer (Hitachi-U-2001, Japan). Each measurement was repeated three times.
Total soluble sugar
Total soluble sugar was measured using the calorimetrical method according to the previous description (Loewus, Reference Loewus1952). Briefly, 0.5 g leaves were ground in 80% ethanol and vortexed followed by incubation for 30 min at 80°C. The mixture was centrifuged at 10,000 rpm for 15 min, the supernatant was mixed with 2 ml anthrone and incubated for 7 min at 80°C. The absorbance was measured at 620 nm and the contents of total soluble sugars were calculated based on the standard curve. Each measurement was repeated three times.
Chlorophylls and carotenoids
We homogenized 0.5 g fresh leaves in 5 ml 80% acetone and filtered the samples using a filter. The absorbance of the homogenate was respectively measured at 645 and 663 nm. The contents of chlorophyll a, chlorophyll b and total chlorophylls were calculated according to the following formulas: chlorophyll a = (12.7 × OD663 − 2.69 × OD645) × V × W/1000, chlorophyll b = (22.9 × OD645 − 4.68 × OD663) × V × W/1000, total chlorophylls = (20.2 × OD645 + 2.69 × OD663) × V × W/1000. Here, ‘V’ means the volume and ‘W’ represents the weight of fresh leaves. The carotenoid contents were determined as described by Arnon (Reference Arnon1949). Each measurement was repeated three times.
Proline
A total of 0.5 g fresh leaves were homogenized with 3% sulfosalicylic acid and filtered with a filter. We mixed 1 ml filtrate with 1 ml 41.67 mg ml−1 ninhydrin solution dissolved in 30 ml glacial acetic acid. The mixture was vortexed and heated at 100°C for 1 h, and the reaction was stopped by ice bath. Then, 4 ml toluene was added and the mixture was incubated for 15–20 min. The absorbance was measured at 520 nm and the proline was estimated based on the previous method (Bates et al., Reference Bates, Waldren and Teare1973). Each measurement was repeated three times.
H2O2 and anthocyanin
A total of 0.5 g fresh leaves were crushed in 5 ml 0.1% trichloroacetic acid (TCA) solution, and the homogenate was transferred into a microfuge tube and then centrifuged at 12,000 rpm for 15 min. A total of 500 μl supernatant was mixed with an equivalent potassium phosphate buffer (pH 7) and 1 ml 1 M potassium iodide solution. We measured the absorbance at 390 and 465 nm. The concentrations of H2O2 and anthocyanin were calculated, respectively, based on the reported method (Velikova et al., Reference Velikova, Yordanov and Edreva2000). Each measurement was repeated three times.
Phenols
A total of 0.5 g leaves were ground in 5 ml 80% acetone and centrifuged at 12,000 rpm for 15 min. A total of 100 μl supernatant was transferred into a 10 ml microfuge tube filled with 1 ml double-distilled water (ddH2O). Then, 2.5 ml Folin–Ciocalteu and 20% Na2CO3 were added and vortexed for 5–10 s. After 20 min of incubation, the absorbance was measured at 750 nm. The standard curve was prepared using the tannic acid method. Total phenol contents were estimated according to the previous description (Julkunen-Tiitto, Reference Julkunen-Tiitto1985). Each measurement was repeated three times.
Malondialdehyde
A total of 0.1 g fresh leaves were collected and homogenized in 1 ml 5% TCA. The homogenate was centrifuged at 12,000 rpm for 15 min. For every aliquot, 1 ml 20% TCA containing 0.5% thiobarbituric acid was added, and the mixture was incubated at 95°C for 30 min. The absorbance was measured at 532 nm. Malondialdehyde was calculated based on the previous method (Heath and Packer, Reference Heath and Packer1968). Each measurement was repeated three times.
Ascorbic acid
A total of 0.25 g leaves were ground in 10 ml of 6% TCA. Then, 4 ml extract was mixed with 2 ml 2% 2,4-dinitrophenyl hydrazine and 10% thiourea solution dissolved in 70% ethanol. The solution was incubated at 100°C for 5 min and then 5 ml of 80% H2SO4 was added. The absorbance was recorded at 530 nm. Each measurement was repeated three times.
Glycine betaine
Glycine betaine was measured according to the description (Di Martino et al., Reference Di Martino, Delfine, Pizzuto, Loreto and Fuggi2003). Briefly, 50 mg of leaves were ground into fine powder in liquid nitrogen followed by a dilution of 2 ml ddH2O. After 24 h of incubation, the homogenate was centrifuged and the supernatant was eluted by High-performance liquid chromatography using a cationic exchange column (Dionex Hypersil SCX, 5 μm, 250 × 4.6 mm) with a flow rate of 1 ml min−1. The eluent phase was 95% 0.05 M sodium phosphate buffer (pH 3.7) and 5% methanol. The eluted glycine betaine was detected by measuring the absorbance of 195 nm and the pure glycine betaine solution was used as a standard.
Enzymatic determination of antioxidant enzymes
For extracting antioxidant enzymes, 0.5 g frozen leaves were ground in 5 ml of 50 mM cooled phosphate buffer. The homogenate was centrifuged at 1500 rpm for 15 min at 4°C and the supernatant was used for enzymatic activity assay.
Superoxide dismutase
A total of 20–50 μl of enzyme extract was mixed with 3 ml of 50 μM nitro-blue tetrazolium, 1.3 μM riboflavin, 13 mM methionine, 75 nm ethylenediaminetetraacetic acid and 50 mM phosphate buffer. The mixture was irradiated under fluorescent lamps at 78 μmol s−1 m−2 for 15 min. The absorbance was read at 560 nm and the activity of SOD was measured according to the previous description (Giannopolitis and Ries, Reference Giannopolitis and Ries1977). Each measurement was repeated three times.
Catalase and peroxidase
A total of 0.1 ml enzyme extract was mixed with a 3 ml catalase reaction solution dissolved in 50 mM phosphate buffer (pH 7.0) and 5.9 mM H2O2. Similarly, 0.1 ml enzyme extract was mixed with 3 ml peroxidase reaction solution dissolved in 50 mM phosphate buffer (pH 5.0), 20 mM guaiacol and 40 mM H2O2. The absorbance was determined at every 20 s, and the activities of catalase and peroxidase were respectively calculated. One-unit catalase or peroxidase activity was defined as the absorbance change per minute. Each measurement was repeated three times.
Data analysis
The attraction rates, amount of egg-laying, hatchability of 11 cultivars (expressed as an average percentage), biochemical substance contents and antioxidase activity data were respectively imported into the statistical computing software Data Processing System v9.50 for analysis (Tang and Zhang, Reference Tang and Zhang2013). All figures were plotted by using GraphPad Prism 7.0 (GraphPad, San Diego, USA).
Results
RPW preferred to infest the Hillawi cultivar
The results obtained from fig. 1a showed that RPW preferred to infest the Hillawi and Mozawati among all 11 date palm cultivars, showing higher attraction rates of 21.66 and 19.13%, respectively and significantly differed from those of Kechanr, Aseel, Khudravi, Shamrani, Dhaki, Zaidi, Zeri, Kobra and Denda. In contrast, the Denda cultivar showed a lowest sensitivity to RPW with a mere 0.43% attraction rate. Among all 11 cultivars, the attraction rates of Mozawati, Kechanr and Aseel were above 10% and those of Khudravi, Shamrani and Dhaki were higher than 5%. In the following, we compared the fecundity and egg hatching rates of RPW fed on these cultivars. Analogous to the above results, the highest amount of egg laying and hatching rate with 17 and 84.67% were respectively recorded in Hillawi (fig. 1b, c). Conversely, RPW did not prefer to lay eggs on the Denda cultivar (average 1.17) and its hatching rate (12.00%) was relatively low, showing less attraction to this pest. The results in this section indicated that RPW preferred to infest the Hillawi and Mozawati cultivars. Considering that the variety Hillawi was the most heavily infested source for weevils and the largest planting cultivar in the Punjab province of Pakistan, the next section, therefore, moved on to discuss the RPW infestation-induced biochemical responses of Hillawi date palm.
Feeding preference of RPW to 11 cultivars. (a) The feeding preference of RPW to 11 cultivars based on two-arm olfactometer assay (n = 40). (b) The number of laying eggs of RPW on 11 cultivars (n = 10). (c) The hatchability of RPW eggs on 11 cultivars (n = 10). The data were analyzed using the Scott Knott test and all statistical differences were marked with different letters (a, b, c, d, e). The results were shown as mean ± SD and each measurement was repeated three times.
RPW infestation did not impair the host photosynthesis
As shown in fig. 2a, RPW infestation did not lead to a remarkable decrease in chlorophyll a content compared to the healthy date palms (P = 0.0558). Also, no significant difference in chlorophyll b content (0.60 μg g−1) was revealed in infected date palms compared with the healthy one (1.12 μg g−1), but we indeed recorded a reduction trend (fig. 2b). Next, we determined the total chlorophyll and carotenoid contents, both showing no obvious changes after RPW infestation (fig. 2c, d). Together these results provided ample evidence that date palms did not impair the capacity of photosynthesis upon RPW infestation.
Comparisons of chlorophyll a (a), chlorophyll b (b), total chlorophylls (c) and carotenoid contents (d) between RPW-infested and healthy date palms. The data were analyzed using an unpaired two-tailed Student's t test and P > 0.05 indicated no statistical significance. The results were shown as mean ± SD and each measurement was repeated three times (n = 3).
RPW infestation increased host antioxidant levels
To figure out the effect of RPW infestation on host anti-oxidative defense responses, we determined the levels of antioxidants in both infested and healthy date palms. Apparently, the contents of these antioxidants including total phenols, proline, H2O2, anthocyanin, malondialdehyde and glycine betaine increased by 1.53–2.48-fold with significant differences except for ascorbic acid content (fig. 3a–f). Among these results, the largest change was observed in proline level, that was, an average of 8.60 μmol g−1 in the infested date palms compared with an average of 3.47 μmol g−1 in healthy plants (fig. 3b). Conversely, we recorded a relatively low fold change in malondialdehyde content, namely, 8.77 μmol g−1 in the infested date palms and 5.74 μmol g−1 in healthy ones (fig. 3e). In summary, RPW infestation remarkably activated the anti-oxidative defense responses of host plants by increasing the antioxidant levels.
Comparisons of total phenols (a), proline (b), H2O2 (c), anthocyanin (d), malondialdehyde (e), ascorbic acid (f) and glycine betaine (g) between RPW-infested and healthy date palms. The data were analyzed using an unpaired two-tailed Student's t test and all statistical significances were marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). The results were shown as mean ± SD and each measurement was repeated three times (n = 3).
RPW infestation increased host antioxidase activity
This section of the survey was concerned with the underlying regulatory mechanism of activated antioxidative defense responses in host plants. Therefore, the activities of three antioxidases were evaluated. As the results presented, the activity of SOD slightly increased by 1.32-fold after RPW infestation, although there was no significant difference (fig. 4a). Analogous to SOD, the RPW-infested date palms did not show a striking increase in peroxidase activity (2.82 units min−1 g−1) compared with healthy ones (2.76 units min−1 g−1) (fig. 4b). However, upon RPW infestation, the enzymatic activity of catalase in infested date palms reached 3.97 units min−1 g−1 relative to the 1.54 units min−1 g−1 in healthy date palms with P < 0.01 (fig. 4c). Overall, these results indicated that the activation of host plant antioxidative defense response was due to the elevation of antioxidase activity.
Comparison of activities of SOD (a), peroxidase (b) and catalase (c) between RPW-infested and healthy date palms. The data were analyzed using an unpaired two-tailed Student's t test and the statistical significance was marked with asterisks (**P < 0.01). The results were shown as mean ± SD and each measurement was repeated three times (n = 3).
RPW infestation elevated host soluble protein and sugar levels
In the final part of this survey, we were concerned about the soluble protein and sugar levels in date palms. The results obtained from fig. 5a showed that total soluble proteins of infested host plants dramatically increased to 7.84 mg g−1, and a lower level of 5.86 mg g−1 was observed in healthy date palms with P < 0.01. In contrast, there was no significant difference in total soluble sugars between RPW-infested (39.67 mg g−1) and healthy hosts (32.33 mg g−1) (fig. 5b).
Comparisons of total soluble proteins (a) and sugars (b) between RPW-infested and healthy date palms. The data were analyzed using an unpaired two-tailed Student's t test and the statistical significance was marked with asterisks (**P < 0.01). The results were shown as mean ± SD and each measurement was repeated three times (n = 3).
Discussion
Herbivorous insects are one of the most speciose groups in invertebrates. They have the capability to infest almost all the greeneries. Accordingly, the attraction of different cultivars to insects varies a lot. In the date palm and RPW model, the preference of RPW to different date palm cultivars has not been figured out. Therefore, the first set of analyses examined the odor preference response of RPW to 11 date palm cultivars, and significant stronger attractions of Hillawi and Mozawati cultivars to RPW were recorded. Further closer inspection showed that RPW prefers to lay eggs on Hillawi and Mozawati in line with the higher hatchability rates. However, considering that the variety Hillawi was the most heavily infested source for weevils and the largest planting commercial cultivar in the Punjab province of Pakistan. Therefore, our subsequent analysis mainly focused on the Hillawi cultivar. Notwithstanding these limitations, these findings are rather encouraging in providing novel insight into the indoor raising of RPW.
Defoliating insects consume mineral contents in the leaves of plants, resulting in the reduction and inhibition of pigment biosynthesis (Jakobs et al., Reference Jakobs, Schweiger and Müller2019). Hence, we compared the contents of photosynthetic pigments in RPW-infested hosts with that of control. Contrary to expectations, the contents of chlorophyll a, chlorophyll b, total chlorophylls and carotenoids did not show remarkably decreases after RPW infestation. Despite we did not record a significant repaired capability in these photosynthetic pigments, the downward trends were quite clear. A previous study demonstrated that these pigments remarkably decreased after infestation by gall forming psyllids (Golan et al., Reference Golan, Rubinowska, Kmieć, Kot, Górska-Drabik, Łagowska and Michałek2015). Also, Moussa et al. (Reference Moussa, Salman and Bakry2012) revealed that the aphid attack caused a serious reduction in host photosynthetic pigment content (Moussa et al., Reference Moussa, Salman and Bakry2012). To further verify the effects of RPW infestation on plant photosynthesis, an important point involving long-term and multipoint detection should be undertaken. Additionally, the transcriptome analysis implied that the debilitating near-term losses in photosynthetic capacity were caused by down-regulation of photosynthesis-related genes (Bilgin et al., Reference Bilgin, Zavala, Zhu, Clough, Ort and Delucia2010). Thus, future studies on this point are therefore recommended.
Our next research depicted an apparent biochemical response of date palms after artificially infested with RPW, showing a significant increase in total phenol contents. Comparison of these findings with the earlier study confirmed the herbivore-medicated antioxidative defensive response correlated with the phenol accumulations (Hasim and Yusuf, Reference Hasim and Yusuf2017). Besides, proline, H2O2, anthocyanin, malondialdehyde, ascorbic acid and glycine betaine are universal non-enzymatic antioxidants not only essential for scavenging ROS, but also modulating many fundamental functions in plants under stress conditions (War et al., Reference War, Paulraj, Ahmad, Buhroo, Hussain, Ignacimuthu and Sharma2012; Akram et al., Reference Akram, Shafiq and Ashraf2017). Our results strongly supported these arguments, showing that these contents were low under normal conditions, but increased in plants after RPW infestation. The potential relationship between significant increased antioxidants induced by herbivorous insects and elevated anti-oxidase activities had been revealed (War et al., Reference War, Paulraj, Ahmad, Buhroo, Hussain, Ignacimuthu and Sharma2012). Thus, we assayed the enzymatic activities of SOD, peroxidase and catalase. In accordance with the above results, the enhancement of SOD, peroxidase and catalase activities was recorded, consistent with other research revealing that psyllid-infested leaves of eucalyptus showed significantly increased SOD and catalase activities (Khattab and Khattab, Reference Khattab and Khattab2005). Taking all the results together, we confirm that RPW infestation activated the host defensive response.
There is growing evidence that the herbivore infestation affects the total soluble protein and sugar levels of host plants (Peumans and Van Damme, Reference Peumans and Van Damme1995; Hogenhout and Bos, Reference Hogenhout and Bos2011). To throw light on this issue, the average level of total soluble proteins between RPW-infested and healthy plants was compared. As expected, there was an elevated level of total soluble proteins after RPW infestation, probably contributing to its defensive role. Besides, it was also well-documented that sugars are involved in plant defense responses under biotic stress (Bolouri Moghaddam and Van den Ende, Reference Bolouri Moghaddam and Van den Ende2012). Therefore, we estimated the total soluble sugars and an increasing trend was recorded in RPW-infested date palms, corresponding to that biotic stress resulted in an increased level of total soluble sugars (Gómez-Ariza et al., Reference Gómez-Ariza, Campo, Rufat, Estopà, Messeguer, Segundo and Coca2007). In contrast, the herbivore-induced concentration of sugars in the leaves of jasmonate biosynthesis-deficient Nicotiana attenuata plants significantly reduced, thus weakening its resistance to Manduca sexta and leading to increased M. sexta growth (Machado et al., Reference Machado, Arce, Ferrieri, Baldwin and Erb2015). However, our results failed to explain the defense mechanism and further research should be undertaken.
Altogether, this research aims to examine the biochemical responses of host date palms to RPW infestation. The most obvious finding was that RPW induced the activation of host antioxidative defense response, complementing those of earlier studies, which indicated that the host plants evolved numerous biochemical and molecular processes to compensate for various environmental damages during the process of mutual confrontation between plants and herbivore insects. These findings, while explorative and preliminary, contribute in several ways to our understanding of plant defense system to resist attackers and provide a basis for breeding insect-resistant crops.
Acknowledgements
We sincerely acknowledge the Higher Education Commission (HEC) of Pakistan for providing a dissertation improvement scholarship (1-8HEC/HRD/2015/5096). This work was funded by the Hainan Provincial Major Science and Technology Project (ZDKJ202002-1.1), the Hainan Provincial Natural Science Foundation of China (320RC473) and the Hainan University Foundation (KYQD(ZR)1963, RZ2100003220).
Conflict of interest
None.
