Resistant cassava cultivars inhibit the papaya mealybug Paracoccus marginatus population based on their interaction: from physiological and biochemical perspectives

Paracoccus marginatus papaya mealybugs cause considerable threats and challenges to cassava production and processing. The deployment of resistant cultivars offers effective, economical and eco-friendly management strategies for pest management. We measured P. marginatus mortality, development and reproduction to evaluate the resistance of fifteen cassava cultivars and conducted physiological and biochemical analyses when P. marginatus was fed on two resistant cultivars (Myanmar and C1115) and three susceptible cultivars (BRA900, Bread, SC205). Significantly lower digestive (amylase, sucrase, lipase), detoxification (glutathione-S-transferase and carboxylesterase) and antioxidant activity, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO), enzyme activities were observed in P. marginatus feeding on resistant cultivars compared to susceptible cultivars. For resistant cultivars, a significant reduction was found in nutritional components containing free amino acids, nitrogen, soluble sugars and the secondary metabolite malondialdehyde. Additionally, significantly higher enzymatic activity (SOD, CAT, POD and PPO) levels and secondary metabolite quantities (total phenol and tannins) were found in resistant cultivars induced by P. marginatus compared with susceptible ones. Additionally, RT-qPCR tests showed that the transcripts of ten genes involved in nutrition, secondary metabolites and antioxidant activities were consistent with physiology changes. Thus, the resistant cultivars suffered lower P. marginatus damage by elevating secondary metabolite contents and antioxidant activities, reducing plant nutrition levels and decreasing insect enzymatic activities. This study will be beneficial in developing indices for standard regulation to evaluate P. marginatus-resistant cassavas and effectively manage this pest.


Introduction
Cassava (Manihot esculenta Crantz), which is known as manioc, is an ancient tropical root and tuber crop that is used for food, feed and industrial production (Parmar et al. 2017). Currently, cassava is widely cultivated in over 100 countries in the tropical and subtropical regions of Africa, America and Asia (Parmar et al. 2017). More importantly, when cassava is treated as a primary foodstuff with high starch content, it fulfils the daily caloric demands of more than 800 million people in developing countries (Parmar et al. 2017). Because of its adaptability to nutrient-poor soil and harsh climatic conditions, easy management and ability to be stored on the ground for long periods of time, cassava is considered a crucial food security crop (Sayre et al. 2011). Nevertheless, some invasive and exotic pests, such as mealybugs, mites and whiteflies, which lead to serious economic damage and yield losses, cause considerable threats and challenges to cassava production worldwide (Bellotti et al. 2012).
Papaya mealybug, Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae), was first described in 1992 from samples collected on cassava in Mexico. Papaya mealybug is a polyphagous insect pest with a host range of more than 200 plants, such as cassava M. esculenta (Malpighiales: Euphorbiaceae), papaya Carica papaya (Parietales: Caricaceae), tomato Solanum lycopersicum (Tubiflorae: Solanaceae) and chili pepper Capsicum annuum (Tubiflorae: Solanaceae) (Finch et al. 2021). Due to human-assisted transport, P. marginatus has been exported rapidly around the world over the last 30 years. Papaya mealybug is currently found in many tropical and subtropical regions of America, Africa, and Asia, including Mexico, Central America, Florida of the USA, Ghana, Tanzania, Kenya, Malaysia, Thailand, India and China (Finch et al. 2021). Infestation by P. marginatus on different host plants can cause crop losses of 10 to 60%, depending on the crop species (Myrick et al. 2014), and even over 90% in some cases (Macharia et al. 2017). Thus, searching for effective strategies to control P. marginatus and aid in the development of agricultural, economic and social benefits is very important.
To avoid damage by insect herbivores, plants have developed many defensive strategies including diverse morphological, biochemical and molecular mechanisms to restrict the effects of herbivore attack (Karban and Baldwin 1997, War et al. 2012;Schuman and Baldwin 2016). These defensive strategies of host plant resistance are commonly classified into constitutive and induced defences (Wu and Baldwin 2010). Moreover, biochemical-based defence (induced defences) in plants may directly affect insect growth, development and survival (Kariyat et al. 2013;War et al. 2018), through the production and accumulation of plant defensive substances, including plant secondary metabolites, oxidative enzymes, proteinase inhibitors and other defensive proteins (War et al. 2012;Züst and Agrawal 2016). The phenolics, flavones and tannins are mainly physiological indicators in the measurement of secondary metabolites in cassava (Yang et al. 2020). Phenols are one of the most common and widespread defensive compounds among plant secondary metabolites and play important roles in defence against insect herbivores and micro-organisms and even against competing plants (War et al. 2012;Singh et al. 2021). For example, flavonol rutin (phenolic compounds) has been reported to have an inhibitory effect on the growth and development of Phenacoccus manihoti (Hemiptera: Pseudococcidae) in cassava plants (Calatayud 2000). Although phenols are constitutively produced in plants, their concentrations are increased in response to insect herbivore attack, which is found in many plants, such as groundnut Arachis hypogaea (Rosales: Papilionoideae) (War et al. 2014) and cotton Gossypium hirsutum (Malvales: Malvaceae) (Dixit et al. 2017). Phenols have direct toxic activity to insects or act as antifeedants (War et al. 2013;Dixit et al. 2017). Tannins, as one group of the most abundant plant secondary metabolites, play an important role in plant defence against phytophagous insects. Previous study reported that tannin content of the cassava was negatively correlated with the population of Prostephanus truncatus (Coleoptera: Bostrichidae), indicating that high tannin content has a restrictive effect on the number of this beetle (Osipitan et al. 2015). Tannins can interact with insect midgut proteins and digestive enzymes to limit their activities in insect pests and ultimately reduce insect growth and development (Barbehenn and Peter Constabel 2011). In addition, the overproduction and rapid accumulation of harmful reactive oxygen species (ROS) in plants are early defence mechanisms in response to arthropods infestation or other abiotic stresses, leading to plant cell and tissue oxidative damage (Mithöfer et al. 2004). To overcome oxidative damage, plants have developed complex and effective enzymatic antioxidant defence systems, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO) and ascorbate peroxidases (APX), to eliminate ROS (Mittler et al. 2004). The roles of these antioxidant enzymes in plant resistance to phytophagous insects have been reported in many plant systems (Erb and Reymond 2019).
On the other hand, arthropods have evolved multiple counter-adaptation mechanisms to overcome the plant defensive system, including morphological, behavioural, biochemical mechanisms (Zhu-Salzman et al. 2005;Birnbaum and Abbot 2018;War et al. 2018). In biochemical traits, digestive enzymes, detoxification enzymes and antioxidant enzymes are considered for insect development and reproduction and play important roles in withstanding plant secondary metabolites, allelochemicals or pesticides (Després et al. 2007;Deng et al. 2013;War et al. 2018). The activities of digestive enzymes such as amylase, sucrase, lipase and proteinase make significant contributions to the adaptive nature of polyphagous herbivores facing various food resources (Zhi et al. 2021). Glutathione-S-transferase (GST), carboxylesterase (CarE) and cytochrome P450-mediated mixed-function oxidase (MFO), as important detoxification metabolism enzymes in insects, can bind and metabolize various endogenous substrates or exogenously applied metabolites (War et al. 2018;Li et al. 2019). In addition, antioxidant enzymes including SOD, CAT, POD and PPO play an important role in defending against the pro-oxidative effects of plant secondary toxic metabolites in insects by eliminating excess ROS (Deng et al. 2013).
Hundreds of cassava genotypes were evaluated by the International Center for Tropical Agriculture (ICTA), and a variety of potential sources of resistance to mite Mononychellus tanajoa (Acari: Tetranychidae), whitefly Aleurotrachelus socialis (Hemiptera: Aleyrodidae) and thrips Frankliniella williamsi (Thysanoptera: Thripidae) were identified (Parsa et al. 2015). In a previous study, the mechanism of resistance to carmine spider mite Tetranychus cinnabarinus (Acari: Tetranychidae) has investigated by comparing morphological characteristics, secondary metabolite defence responses and proteomic analysis in different cassava genotypes (Yang et al. 2020). Recently, the resistance of multiple cassava cultivars to T. cinnabarinus was confirmed via laboratory and field evaluations, and cassava cultivars BRA900 and C1115 were defined as susceptible and resistant cultivars, respectively ). However, little is known about cassava cultivars in response to P. marginatus. In addition, this notorious pest has the characteristics of quick development and high fecundity (Amarasekare et al. 2008), which gives it great potential to adapt to new areas and reach high population densities in the short term. Hence, it is important and meaningful to identify resistant cassava cultivars to P. marginatus. Here, we selected fifteen cassava cultivars to evaluate their susceptibility and resistance to P. marginatus. To understand why resistant cultivars inhibit P. marginatus, the activity changes of digestive enzymes, detoxification enzymes and antioxidant enzymes in P. marginatus were tested. In addition, the effects on the contents of nutrients and secondary metabolites, antioxidant enzyme activities and mRNA levels were measured in susceptible and resistant cassava cultivars after P. marginatus infestation. This study is helpful to understand cassava resistance to P. marginatus and will contribute to establishing standard regulations to evaluate P. marginatus-resistant cassava germplasms and help in the development of effective strategies for P. marginatus management.

Cultivation of cassava cultivars
Fifteen cassava cultivars, BRA900, Bread, Nuomi, ZM9066, Swiss T7, SC205, GR5, SC6068, J1301, DG hongwei, SC11, SC8002, SC8, Myanmar and C1115, were supplied by the National Cassava Germplasm Nursery of China, Chinese Academy of Tropical Agricultural Sciences (CATAS). The cassava cultivars BRA900 and C1115 functioned as susceptible and resistant cultivars, respectively . All cassava cultivars were cultivated in pots (33 cm in diameter, 25 cm in height) with 5 kg of well-mixed soil (soil: peat: perlite = 1:1:1) and were grown in an insect-free greenhouse at 28 ± 1 °C with a 14:10 h (light/dark) photoperiod. Three months later, these plants were used to test the effects of P. marginatus on development and reproduction or for enzyme activity assays (details below).

Insect rearing
The P. marginatus colony was originally collected from cassava fields in Danzhou city, Hainan Province, China (19°30′44″N, 109°29′26″E), in 2017. It was continuously maintained on susceptible BRA900 plant for numerous generations at the Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences under the following conditions: temperature 28 ± 1 °C, 75 ± 5% relative humidity (RH), and a 14:10 h (L/D) photoperiod in a temperature-controlled greenhouse. Similar growth status leaves were used to rear P. marginatus from 10-16 leaves below the apical bud of a 3-month-old BRA900 cultivar.

Mortality evaluation of P. marginatus feeding on BRA900 and C1115
Mortality was evaluated, while P. marginatus fed on either BRA900 or C1115, a cassava cultivar which was susceptible and resistant to mite, respectively ). The 1 3 idea is to confirm whether these two cultivars also exhibit similar resistance pattern during mealybug infestation. The cassava leaf from middle to basal was picked and placed with the surface on a layer of foam dampened with deionized water, and the leaf margin was surrounded with a watersaturated blotting paper strip to prevent the papaya mealybug from escaping and to keep the leaf fresh. After third-instar nymphs molted and transformed into adults, thirty 1-dayold female adults were carefully inoculated on the back of cassava leaf from 3-month-old plants using a fine Chinese writing brush. In addition, the cassava leaf was replaced every two days. The survival was recorded every day for a total period of 10 days.

The effects on mortality, development and reproduction of P. marginatus on different cassava cultivars
To evaluate the influence of 15 cassava cultivars on the mortality, development and reproduction of P. marginatus, thirty 1-day-old female adults were fed on the backs of a leaf of each cassava cultivar. Three leaves from middle to basal part of one plant were used. The mortality of female adults was assessed on days 1 and 4 based on the results of the above experiments. In addition, the mature female adults were allowed to lay eggs for 24 h after inoculation, and the eggs remained near. After eggs hatching, individual larva was placed in the sections divided by the water-saturated blotting paper strip on the surface of each cassava cultivar leaf for development observation. A leaf was divided into six sections. The developmental duration of eggs, first-to third-instar nymphs and female adults of F 0 P. marginatus were recorded every 12 h. For each developmental stage, at least 20 tested insects were observed per cassava cultivar. Furthermore, one leaf with a female adult from each of fifteen cassava cultivars was used to assess the fecundity and egg hatchability of the P. marginatus until the adult died. The mean eggs per female and egg hatchability of F 1 P. marginatus were recorded using a microscope. Twenty individuals were divided into three biological replicate groups and observed for each cassava cultivar (n = 6-7).

P. marginatus collection for enzyme solution
Based on the results of mortality, development and reproduction of P. marginatus on different cassava cultivars, the effects of diverse enzyme activities in P. marginatus were measured from the following cassava cultivars: BRA900, Bread, SC205, Myanmar and C1115. Among them, BRA900 and C1115 were used as the mealybug-susceptible control and mealybug-resistant control, respectively. For each cassava cultivar, many 1-day-old female adults were placed on the back of the leaf, and 30 surviving mealybugs (~ 0.025 g) were collected for enzyme extraction after one day and four days. The samples were ground in liquid nitrogen and thoroughly homogenized in distilled water before undergoing centrifuging at 7,000 rpm for 15 min at 4 °C. The supernatant was collected and frozen at -80 °C until enzyme activity assay. Each treatment included three independent biological replicates.

Measurement of digestive enzyme activity in P. marginatus
Amylase activity was determined following a method previously described (Vatanparast and Hosseininaveh 2010) with slight modification. In brief, 20 μL enzyme extract and 100 μL phosphate buffer (0.2 M, pH = 5.8) and 50 μL of 2% soluble starch were incubated for 30 min at 35 °C. Then, the mixed solution was terminated by the addition of 250 μL dinitrosalicylic acid (DNS, Sigma-Aldrich, US) and heating in boiling water for 5 min. After cooling, the absorbance of the reaction mixture was detected at 550 nm using a microplate reader Spark 20 M (Tecan, Switzerland). In the blanks, phosphate buffer was used instead of enzyme extract. Maltose was used to generate the standard curve. All assays were conducted in triplicate.
Sucrase activity was measured following a previous study with some minor modifications (Karley et al. 2005). Enzyme extract (20 μL) was mixed with 100 μL phosphate buffer (0.2 M, pH = 6.5) and 50 μL 1% sucrose solution for 10 min at 35 °C. The termination and absorbance measurement of the reaction were taken as described above. The glucose was used to generate the standard curve. The measurement was repeated three times.
Lipase activity was determined as previously described (Choi et al. 2003) with a minor modification. The standard reaction mixture (pH = 7.5) was composed of 40 mM 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), 10 mM 2,3-dimercapto-1-propanol tributyrate (DMPTB), 0.5 M ethylenediaminetetraacetic acid, 10% Triton X-100, and 1 M Tris-Cl. A volume of 20 μL enzyme sample was added to 180 μL of this mixture in 96-well microtiter plates and incubated at 37 °C for 30 min. The absorbance variation was measured at 405 nm. We used a blank that had no DMPTB, and triglycerides were used to generate the standard curve. Three independent biological replicates were performed for each reaction.

Measurement of detoxification enzyme activity in P. marginatus
Carboxylesterase (CarE) activity was determined as previously reported (Asperen 1962) with some modifications. Briefly, 75 μL enzyme sample was added to 100 μL substrate with 1 × 10 -4 M eserine and 3 × 10 -4 M α-naphthyl acetate (α-NA) in phosphate buffer (0.04 M, pH = 7.0). After incubating for 10 min, 25 μL of the fast blue conjugate dye was added to the reaction mixture for 10 min at 30 °C. The assay was carried out in 96-well microtiter plates, and the absorbance was measured at 600 nm using a microplate reader. In the control, phosphate buffer was used instead of enzyme sample. α-NA was used to generate the standard curve. All assays were conducted in triplicate.
Glutathione S-transferase (GST) activity was determined following a method previously described (Habig et al. 1974) with some modifications. Briefly, 1-chloro 2-4,dinitrobenzene (CDNB) was used as the substrate. A 200 μL mixture of 0.6 mM CDNB and 6 mM reduced glutathione (GSH) was pipetted into 96-well microtiter plates and was incubated at 37 °C for 20 min. Then, 80 μL of enzyme sample was added to the reaction mixture, and the absorbance at 340 nm was recorded every 30 s in 30 min. In the control, the same volume of phosphate buffer (0.04 M, pH = 7.0) was used instead of enzyme sample. The GSH was used to generate the standard curve. Three independent biological replicates were evaluated for each reaction.
Mixed-function oxidase (MFO) activity was measured using a modified method . In brief, an aliquot of 100 μL enzyme sample was added to 50 μL substrate p-nitroanisole (1 mM) and was incubated at 37 °C for 5 min. Thereafter, the mixture was mixed with 50 μL nicotinamide adenine dinucleotide phosphate (NADPH, 1 mM), and the absorbance was estimated at 450 nm. The reaction mixture without the enzyme extract was treated as a blank control. p-Nitrophenol was used to generate the standard curve. The measurement was repeated three times.

Measurement of the antioxidant enzyme activity in P. marginatus
Activities of SOD, CAT, POD and PPO were assayed according to a previous description (Lu et al. 2016) with minor modifications. The absorbance for SOD, CAT, POD and PPO assay was estimated at 325, 415, 470 and 398 nm. All experiments were performed in triplicate.

Cassava sample collection and the determination of antioxidant enzyme activity
To evaluate the physiological and biochemical responses of five cassava cultivars (BRA900, Bread, SC205, Myanmar and C1115), thirty 1-day-old female adults of P. marginatus were placed on the back of leaves from 3-month-old plants.
Based on the results of the above experiments, the adults were removed after inoculation for 0, 1 and 4 d, and then, 100 mg of damaged leaves without petiole were collected at the same time points. The plant samples were ground into powder with a mortar and pestle and thoroughly triturated in extract solution before centrifugation at 7,000 rpm for 15 min at 4 °C. Each insect-damaged leaf was treated as a biological replicate, and each cassava cultivar treatment contained three leaves. Additionally, the antioxidant enzyme activities of SOD, CAT, POD and PPO in cassava plants were measured as described above.

Determination of the nutrient substance in M. esculenta
In each cassava cultivar treatment, 100 mg was extracted with 10 mL 80% ethanol and was incubated in a water bath at 80 °C for 20 min. Then, the extraction was centrifuged at 5,000 g for 15 min, and the supernatant was used to determine the content of soluble sugar (SS) and free amino acids (FAA) after reacting with anthrone and ninhydrin, respectively (Yemm and Willis 1954;Yemm et al. 1955;Cao et al. 2008). The absorbance was estimated at 620 and 570 nm, and glucose and alanine were used as standards. The analysis of free proline content was carried out following a previous method with some modifications (Gibon et al. 2000;Karalija and Selović 2018). In brief, 100 μL of each supernatant was mixed with 1 mL of reaction mixture (1% ninhydrin, 60% acetic acid, and 20% ethanol) and heated at 95 °C for 20 min. After cooling, the chromogen that formed was extracted with 3 mL toluene and separated at 3,000 g for 5 min. The supernatant was used to estimate the absorbance at 520 nm, and L-proline was used as a standard. The soluble nitrogen (N) content of each sample was determined using Coomassie brilliant blue at a wavelength of 595 nm. All experiments were performed with three biological replicates.

Determination of the secondary metabolites in M. esculenta
Total phenol content was determined following the spectrophotometric method previously described (Maisetta et al. 2019) with some modifications. In short, 1 mL of each cassava sample was mixed with Folin-Ciocalteu reagent (0.25 M) and was incubated at 25 °C for 3 min. Then, a 1-h incubation in the dark at 25 °C with 1 mL Na 2 CO 3 (10%, w/v) solution followed. The absorbance was read at 760 nm, and gallic acid was used as a standard to perform the calibration. The method used to determine tannin content was similar to the method of total phenol content described above. For the determination of malondialdehyde (MDA), a mixture of 1 mL of each cassava sample and 2 mL 0.25% thiobarbituric acid (TBA, w/v) in 10% trichloroacetic acid (TCA, w/v) was boiled for 15 min, quickly cooled on ice and then centrifuged at 10,000 g for 20 min. The supernatant was used to estimate the absorbance at 600, 532 and 450 nm following a formula previously described (Wei et al. 2013). All the experiments were performed in triplicate.

RNA extraction and quantitative real-time PCR (qPCR)
This study aims to elucidate the mealybug-resistant mechanism of cassava based on three aspects including nutrition substrate abundances, secondary metabolite contents and antioxidant enzyme activities. In addition, these physiological and biochemical characteristics were potentially regulated by specific genes. Thus, ten genes including five nutrient synthesis genes (nitrate reductase, NR; glutamine synthetase, GS; sucrose synthetase, Susy; pyrrolin-5-carboxylic acid synthetase, P5CS; ornithine aminotransferase, OAT), two tannin synthesis genes (leucoanthocyanidin reductase, LAR; anthocyanidin reductase, ANR) as well as three antioxidant enzyme encoding related genes (POD, CAT , Cu/ZnSOD) were subjected to transcription analysis. Ten specific genes for RT-qPCR were designed using Primer-BLAST software (https:// www. ncbi. nlm. nih. gov/ tools/ prime rblast) and are shown in Table S1. Total RNA was extracted from the leaves of three susceptible cultivars (BRA900, Bread and SC205) and two resistant ones (Myanmar and C1115) after inoculation with mealybugs collected at 0, 1 and 4 d. Each treatment and cultivar included three biological replicates. For each damaged leaf (~ 0.1 g), total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), the integrity was verified by 1% agarose gel electrophoresis, and the quantity was evaluated using a NanoDrop spectrophotometer 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Next, 1 μg RNA was converted to first-strand complementary DNA using an RT EasyMix Kit with gDNA Eraser (Tolo Bio, Shanghai, China) in accordance with the manufacturer's instructions. An ABI StepOnePlus Real-Time PCR System (PerkinElmer Applied Biosystems, CA, USA) was used to analyse the tested gene transcripts with SYBR qPCR Master mix (Tolo Bio). The RT-qPCR program was as follows: denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Melting curve analysis (60-95 °C) was used to validate the specificity of each primer. Cassava Actin (KM583807.1) was used as a reference, and the relative quantification was calculated based on the comparative 2 −ΔΔCt method (Livak and Schmittgen 2001). Three biological replicates and three technical replicates were employed to detect gene expression levels.

Statistical analyses
All data were statistically analysed using GraphPad Prism software 8.0 (GraphPad Software, San Diego, CA, USA) and Statistical software SPSS 26.0 (SPSS Inc., Chicago, IL, USA). For the P. marginatus experiment mortality evaluation, the log-rank (Mantel-Cox) test was used to analyse the significant difference. The mortality, development and reproduction of P. marginatus experiments, various enzymatic activities, the contents of nutrition compositions and secondary metabolites among different cultivars, and gene transcript levels were compared by one-way analysis of variance (ANOVA) with Tukey's honestly significant difference (HSD) multiple comparison test. Additionally, the changes in nutrition compositions, secondary metabolites and antioxidant enzyme activities within cassava were analysed using Student's t-test. All data are shown as the means ± standard errors (SE), and a P value less than 0.05 was considered statistically significant.

Mortality analysis of P. marginatus on BRA900 and C1115
There was a significant difference in mortality of female adults between the susceptible cultivar BRA900 and resistant cultivar C1115 (Fig. 1). From the first day, the difference became more obvious. The mortality of female adults feeding on C1115 increased from 13.33 to 100% on days 1-10. However, the changes in mortality of the BRA900 group maintained a slow increasing trend in the 10-day period (3.33 to 53.33%). Significantly, the mortality of the C1115 group increased to 50%, with higher variation on the fourth day compared to that of the BRA900 group (χ 2 = 22.61, df = 1, P < 0.001). Therefore, these results suggest that the first and fourth days were important timepoints for P. marginatus responding to susceptible and resistant cassava cultivars. Fig. 1 Mortality of P. marginatus after feeding on BRA900 and C115 cultivars. Thirty 1-day-old female adults were included in each treatment, and their mortality was evaluated. The difference in mortality curves was analysed using the log-rank (Mantel-Cox) test (***P < 0.001)

The mortality, development and reproduction of P. marginatus assay in different cassava cultivars
To investigate the influence of 15 cassava cultivars on P. marginatus, the mortality, development and reproduction of P. marginatus were evaluated. The results showed that the average mortality of female adults (F 0 ) was significantly different (1 d, F = 14.03, df = 14, P < 0.001; 4 d, F = 57.48, df = 14, P < 0.001) among the 15 cassava cultivars ( Fig. 2A). Based on the mortality assay at 1 and 4 d, the resistance of different cassava cultivars responding to P. marginatus was ranked as follows: BRA900 (susceptible control), Bread, SC205, ZM9066, Swiss T7, Nuomi, SC6068, GR5 < J1301, DG hongwei, SC11, SC8002 < SC8, Myanmar and C1115 (resistant control). Furthermore, the developmental duration of P. marginatus feeding on different cassava cultivars was evaluated, including eggs, first-third nymphs and female adults. Significant differences in developmental duration were observed in the egg, first and third nymph stages (egg, F = 6.86, df = 14, P < 0.001; 1 st nymph, F = 96.73, df = 14, P < 0.001; 3 rd nymph, F = 19.95, df = 14, P < 0.001), but no significant changes in the second nymph stage were seen in 15 cassava cultivars (F = 0.79, df = 14, P = 0.67) (Fig. 2B). Overall, the whole developmental period (egg to adult) of P. marginatus feeding on resistant cassava cultivars was significantly longer than the duration in susceptible cultivars (F = 32.86, df = 14, P < 0.001). In addition, there were significant differences in the total eggs laid per female (F 1 ) after feeding on different cassava cultivars (F = 62.91, df = 14, P < 0.001) (Fig. 2C). The average number of eggs per female in eight cassava cultivars (BRA900, Bread, SC205, ZM9066, Swiss T7, Nuomi, SC6068 and GR5) was 367.6 (± 7.6, SE), which was significantly higher than that in the six resistant cassava cultivars, including J1301, DG hongwei, SC11, SC8002, SC8, Myanmar and C1115 (average 141.6 ± 9.5 eggs). The average egg hatchability of eight cassava cultivars was 98.4%, which was significantly Fig. 2 Effects of different cassava cultivars on the mortality, development and reproduction of P. marginatus. A, Mortality assay of F 0 female adults feeding on 15 cassava cultivars on days 1 and 4. Thirty individuals were used in each treatment. B, Developmental duration (egg to female adult) of P. marginatus (F 1 ) feeding on 15 cassava cultivars. C, Determination of the total number of eggs laid per female and D, hatchability of P. marginatus. Twenty individuals were used for each cassava cultivar. Different letters on the standard error (SE) bars represent statistically significant differences among different cassava cultivars using one-way ANOVA followed by Tukey's honestly significant difference (HSD) test (P < 0.05) higher than the hatchability of the remaining seven cassava cultivars (47.1%) (Fig. 2D).

Activity of digestive enzymes in P. marginatus feeding on cassava plants
To illustrate what physiological responses occurred in P. marginatus feeding on susceptible and resistant cassava plants, the activity of digestive enzymes of P. marginatus was first measured. As shown in Fig. 3A, the amylase activity of P. marginatus feeding on Myanmar and C1115 for 1 and 4 d was 22.37, 21.34 and 24.67, 20.49 U/L, respectively, which was significantly lower than that in BRA900

Fig. 3
Changes in digestive enzyme activities in P. marginatus feeding on susceptible and resistant cassavas. A, Amylase activity; B, sucrase activity; C, lipase activity. Thirty papaya mealybugs were used for each treatment. All the data shown are the means of enzyme activities ± SE (n = 3), and different letters on the bar indicate a significant difference in ANOVA (HSD, P < 0.05) Fig. 4 Changes in detoxification enzyme activities in P. marginatus feeding on susceptible and resistant cassavas. A, Carboxylesterase (CarE) activity; B, glutathione S-transferase (GST) activity; C, mixed-function oxidase (MFO) activity. Thirty papaya mealybugs were used for each treatment. The vertical bars represent the SE of the mean over three biological replicates. Different letters above each bar represent the significant differences in detoxification enzyme activities among different cassavas determined by one-way ANOVA (HSD, P < 0.05)

Fig. 5
Changes in antioxidant enzyme activities in P. marginatus feeding on susceptible and resistant cassavas. A, superoxide dismutase (SOD) activity; B, catalase (CAT) activity; C, peroxidase (POD) activity; D, polyphenol oxidase (PPO) activity. Thirty 1-day-old female papaya mealybugs were used for each treatment. The vertical bars are SE of the mean (n = 3). Different letters indicate statistically significant differences among different cassavas using one-way ANOVA (HSD, P < 0.05)

Activity of antioxidant enzymes in P. marginatus feeding on cassava plants
The activities of antioxidant enzymes (SOD, CAT, POD and PPO) in P. marginatus were differently affected by inoculation on susceptible and resistant cassavas (Fig. 5). The results showed that significantly lower levels of SOD, CAT, POD and PPO activities were detected in two resistant cassava plants than in three susceptible cassava plants

Content analysis of the secondary metabolites in M. esculenta
The total phenol and tannin contents of cassava leaves damaged by P. marginatus from susceptible and resistant cultivars are shown in Table 1. The total phenol (1 d, F = 10.20, Fig. 6 Changes in nutritional composition in M. esculenta damaged by P. marginatus. A, Soluble sugar (SS); B, nitrogen (N); C, free amino acid (FAA); D, free proline. The vertical bars indicate the SE of the mean over three biological replicates. Different letters represent significant differences in nutrient contents among different cassava cultivars during the same time using one-way ANOVA (HSD, P < 0.05). In addition, leaves without mealybug infestation at 0 d were set as the control group. The asterisk indicates significant differences with a cassava among different times using Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant differences) df = 4, P = 0.001; 4 d, F = 14.09, df = 4, P < 0.001) and tannin (1 d, F = 80.91, df = 4, P < 0.001; 4 d, F = 118.50, df = 4, P < 0.001) contents in resistant cassavas were significantly higher than those in susceptible cassavas at 1 and 4 d.

Discussion
Development and reproduction are crucial factors in the adaptation of pests to host plants (Lu et al. 2016;Zhang et al. 2021). Previous studies reported that herbivorous insects respond to host variations not only among different plant species, but also among different cultivars of the same plant variety (Lu et al. 2016). For example, the fecundity, hatchability, developmental duration and lifespan of T. cinnabarinus on transgenic cassava lines SC2, SC4 and SC11 overproducing MeCu/ZnSOD and MeCAT1 were significantly lower than those on wild-type cultivar TMS60444 . Similarly, there were significant differences in the growth, development and reproduction of papaya mealybugs fed on different cassava cultivars. Based on our results, BRA900, Bread and SC205 can be identified as susceptible to P. marginatus, whereas SC8, Myanmar and C1115 were identified as resistant to P. marginatus. The mortality, egg hatchability, developmental duration and fecundity of P. marginatus feeding on resistant cassava cultivars were significantly restricted, suggesting that these can be used as important indices to evaluate the susceptibility and resistance of different cassava cultivars to P. marginatus.
The nutrition levels and allelochemicals in plants are closely related to plant suitability and resistance to herbivorous insects (Chen et al. 2009). We found that the contents of FAA, N and SS in resistant cassava cultivars were significantly lower than those in susceptible cassava cultivars post P. marginatus infestation, suggesting that resistant cassava cultivars may decrease nutrition levels to response to mealybug infestation. Amino acid nutrition is critical for insect development. For example, the fecundity and growth rate of Rhopalosiphum padi (Hemiptera: Aphididae) are directly proportional to the free amino acid (FAA) levels in its host plant (Weibull et al. 1990). Thus, a lower FAA content in resistant cassava cultivars limits the development of P. marginatus. In contrast, the content of FAA in susceptible cassava cultivars acting as suitable plants significantly increased in response to P. marginatus damage. Similar results were also found in Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), infested Satsuma orange leaves (Malik et al. 2014). Constituents of host plant quality, such as sugar and nitrogen (N), play a determinant role in the development and fecundity of insect herbivores (Awmack and Leather 2002).

Table 2
Changes in the antioxidant enzyme activity in M. esculenta damaged by P. marginatus The value (mean ± SE) is presented as the mean of three biological replicates. Different letters indicate significant differences in antioxidant enzyme activity within a column using one-way ANOVA (HSD, P < 0.05). Leaves without mealybug infestation at 0 d were set as the control group. The asterisk in a row indicates significant differences using Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant differences) Cultivars SOD (U/mL) Our results showed that resistant cassava cultivars inhibit the development and fecundity of P. marginatus likely by reducing N and SS contents, while P. marginatus infestation results in a significant increase in the nutritional composition of susceptible cassava. Furthermore, the mRNA transcription level changes of two genes, MeNR and MeGS, involved in N metabolism and one gene, MeSusy, involved in sucrose synthesis were consistent with the changes in nutrient contents. This indicated that the observed reduction in nutrition levels may be due to the decrease in mRNA expression in resistant cassava cultivars. In addition, proline is known to be involved in the adaptation of plants in response to biotic and abiotic stresses, such as insect herbivore attack, pathogen infection and drought (Yang et al. 2011;Qamar et al. 2015;Kang et al. 2018). In our study, the free proline content in susceptible and resistant cassava plants was significantly increased after P. marginatus attack, and the expression of two genes, MeP5CS and MeOAT, in relation to proline synthesis also were significantly upregulated in both the susceptible and resistant cassava plants. The P5CS gene is generally used in metabolic engineering for proline excessive accumulation conferring biotic and abiotic stress tolerance in plants (Nai and Penna 2013). Thus, the transcript and free proline accumulation in P. marginatus-damaged cassava indicated that the plant defensive mechanism was triggered.
To counter insect herbivore feeding, plants have developed a series of defence strategies, including direct and indirect, constitutive and induced, physical and chemical defences (secondary metabolites), which are a critical group that contributes to plant defence against insect attack (War et al. 2012;Schuman and Baldwin 2016;Singh et al. 2021). Among the secondary metabolites, plant phenols are one of the most important compounds against insect pests (War et al. 2012). For instance, the resistant cassava cultivar XX048 to T. cinnabarinus was significantly enriched in the biosynthesis of flavonoids (phenolic compounds) compared to susceptible cultivar GR4 via proteomic and transcript analysis (Yang et al. 2020). Our results showed data are the mean ± SE of three biological replicates. Cassava Actin (KM583807.1) was used as a reference gene, and the relative transcripts were analysed using the comparative 2 −ΔΔCt method. Lowercase letters above the bars represent significant differences in expression levels among different times using one-way ANOVA (HSD, P < 0.05) that a significantly higher total phenol content was found in resistant cassava plants following P. marginatus infestation than in susceptible cassava plants. This suggested that the increased content of total phenol in resistant cassava may negatively affected growth, development and fecundity in P. marginatus. Similar results were reported in a previous study, which found a higher level of phenolic compounds in resistant winter triticale responding to Sitobion avenae (Hemiptera: Aphididae) and Oulema melanopus (Coleoptera: Crioceridae) infestation than in susceptible winter triticale (Czerniewicz et al. 2017). Many similar examples in connection with phenolic compounds defence against herbivorous insects were documented by Singh et al. (2021).
Qualitative and quantitative changes in phenols and enhancement of the activities of oxidative enzymes against herbivorous insects are common defences (Maffei et al. 2007;War et al. 2011;Czerniewicz et al. 2017). For example, higher levels of various antioxidant enzyme activities and contents of secondary metabolites, including phenols, were found in insect-resistant groundnut genotypes pretreated with jasmonic acid and then damaged by Helicoverpa armigera (Lepidoptera: Noctuidae) compared with susceptible genotypes (War et al. 2014). In this study, the transcripts (MeCu/ZnSOD, MeCAT and MePOD) and activities of antioxidant enzymes (SOD, CAT, POD and PPO) in resistant cassava were significantly elevated following P. marginatus infestation compared with susceptible cassava. Similarly, significant upregulation of mRNA levels together with activities of SOD, CAT, POD and PPO was also observed in mite-resistant cassavas in response to T. cinnabarinus and T. urticae feeding but not susceptible Lu et al. 2017). These results indicated that phenols are involved in the cyclic reduction of ROS, which in turn activates a set of reactions, resulting in the activation of antioxidant enzymes to defend against insect pest attack (Maffei et al. 2007). The activities of antioxidant enzymes were determined in P. marginatus and significantly lower enzymatic activities were found in P. marginatus feeding on resistant cassava compared to those in susceptible cassava. The reduction in the activities of antioxidant enzymes in insect herbivores can contribute to host plant resistance (War et al. 2014). For example, previous studies reported that the downregulation of endogenous protective enzyme activities significantly inhibited the survival, development and production of Eotetranychus sexmaculatus (Acari: Tetranychidae) (Lu et al. 2016) and T. cinnabarinus (Acari: Tetranychidae) ) after feeding on resistant rubber trees and cassava, which may reduce the self-protection capacity of pests and contribute to plant resistance to pests. The activity analysis of antioxidant enzymes in plants and pests aids in understanding their interaction, and our findings showed that resistant cassava plants can elevate the total phenol content as well as antioxidant enzyme activities to defend P. marginatus. Our results suggested the potential importance of upregulated total phenol content and antioxidant enzyme activities as key indices to judge cassava resistance to P. marginatus.
Plant tannins, polyphenolic plant secondary metabolites, are involved in plant defence against insect herbivores by deterrence and/or toxicity (Barbehenn and Peter Constabel 2011). In the present study, resistant cassavas presented significantly higher tannin contents in response to P. marginatus damage than susceptible cassavas. Similarly, high tannin content of cassava chips has a negative effect on adult P. truncatus population, suggesting that the relative resistance of cassava varieties to damage by P. truncatus may likely be conferred by this secondary metabolite (Osipitan et al. 2015). Our results also showed that the transcript levels of MeLAR and MeANR were higher in resistant cassava induced by P. marginatus than in susceptible cassavas. This indicated that the observed accumulation of tannins may be because of the upregulation of mRNA transcript levels in resistant cassavas. In addition, the induction of tannins in plant defence against insect damage and their influence on insect management have been studied in various plants, such as Pinus sylvestris (Pinales: Pinaceae) (Roitto et al. 2009) and A. hypogaea (Rosales: Papilionoideae) (War et al. 2014). Plant tannins are protease inhibitors that bind to digestive enzymes to form a complex, precipitate proteins nonspecifically and reduce the digestion and utilization of plant nutritive value to insect herbivores (Barbehenn and Peter Constabel 2011). During the growth and development of insects, digestive enzymes play an important role in the digestion and absorption of nutrients. We found that the activities of amylase, lipase and sucrase were significantly inhibited in P. marginatus after they were fed on resistant cassavas, which, may probably the accumulation of tannins in resistant cassavas, caused the inhibition of digestive enzymes in P. marginatus. Similarly, a significant reduction in the activities of amylase and lipase was observed in Lymantria dispar (Lepidoptera: Lymantriidae) larvae under the secondary metabolite carvacrol (terpene compound) treatment (Chen et al. 2021). In addition, digestive enzyme (amylase and trypsin) activities were reduced in both nymphs and adults of Frankliniella occidentalis (Thysanoptera: Thripidae) when they were transferred from the more preferred host kidney bean to a less-preferred host broad bean, potentially leading to reduced fitness of F. occidentalis (Zhi et al. 2021). These results imply that different plant species or different cultivars of the same plant species with different plant secondary metabolite contents could affect insect fitness by changing its digestive capacity.
Malondialdehyde (MDA) content is considered a key parameter of oxidative damage in the plant cell membrane in response to biotic and abiotic stress, and many studies have used MDA content to evaluate the extent of plant defence against insect herbivore attack (Wei et al. 2007;Hao et al. 2011;War et al. 2011War et al. , 2014. In addition, ROS may induce lipid peroxidation in the membrane and result in the formation of lipid peroxidation products such as MDA. In this study, our findings revealed that susceptible cassavas showed higher MDA content than resistant cassavas after P. marginatus infestation. Similar results were also found in different alfalfa varieties in which susceptible varieties always presented higher MDA contents than resistant varieties when they were pierced and sucked by Aphis medicaginis (Hemiptera: Aphididae) (Wei et al. 2007). Higher amounts of MDA in susceptible groundnut in response to Spodoptera litura (Lepidoptera: Noctuidae) infestation have been well documented in a previous study (War et al. 2011). These results indicated that the cellular membrane systems in susceptible plants suffer much more serious damage than those in resistant plants.
Insects detoxify plant toxic secondary metabolites through many detoxification enzymes, such as GST, CarE and P450 (War et al. 2018;Li et al. 2019). For example, S. avenae developed increased activities of GST and CarE to defend against the plant secondary metabolite gramine, and they were positively correlated with the concentration of dietary gramine (Cai et al. 2009). A previous study suggested that tannic acid elevated GST, CarE and P450 activities in Hyphantria cunea (Lepidoptera: Arctiidae) larvae in a concentration-dependent and time-dependent manner (Yuan et al. 2020). However, it was shown that tannic acid and quercetin could decrease the activity of GST in two moth species in a concentration-dependent manner (Tang et al. 2014). In this study, we found that the CarE and GST activities of P. marginatus were significantly inhibited in resistant cassavas, suggesting that the increase of plant secondary metabolites such as phenols and tannins in resistant cassavas resulted in the reduction of detoxification enzyme activities (CarE and GST) of P. marginatus. Similar to the response of L. dispar larvae to carvacrol, the activities of CarE, GST and acetylcholinesterase (AchE) were significantly decreased (Chen et al. 2021). In addition, significant upregulation of MFO activity of P. marginatus was observed in resistant cassavas compared with susceptible cassavas, implying that P. marginatus may have developed different physiological adaptations to plant secondary metabolites.

Conclusion
Resistance analysis of fifteen cassava cultivars from CATAS was performed and characterized by the evaluation of the mortality, development and reproduction of P. marginatus. The mechanisms of cassava resistance to P. marginatus in physiological and biochemical responses were evaluated from the perspectives of both the plant and the insect and are summarized in Fig. 8. Our results revealed that higher levels of toxic secondary metabolites (total phenol and tannins) together with antioxidant enzyme activities (CAT, Fig. 8 Summary of the induced responses in cassava and P. marginatus. Resistant cassavas are unsuitable for P. marginatus adaptation because plants enhance the levels of toxic secondary metabolites as well as the activities of antioxidant enzymes and weaken nutritional compositions in response to P. marginatus infestation. The plant gene changes induced by P. marginatus are consistent with the changes in nutrition, secondary metabolites and enzymatic activities. Thus, unsuitable plants inhibit the activities of digestive enzymes, detoxification enzymes and antioxidant enzymes and ultimately result in poor population levels of P. marginatus and vice versa in susceptible (suitable) cassavas. The red arrow represents upregulation, and the green arrow indicates downregulation SOD, POD and PPO) and lower contents of nutrients (SS, N, FAA and proline) and MDA were observed in resistant cassava cultivars infested with P. marginatus. In addition, the mRNA transcription level variations of these related genes were consistent with the changes in nutrition, secondary metabolites and enzymatic activities. The activities of digestive enzymes (amylase, sucrase and lipase), detoxification enzymes (CarE and GST) and antioxidant enzymes of P. marginatus were significantly inhibited after they were fed on resistant cultivars. Therefore, the resistant cassavas suffered lower pest damage and led to poor survival as well as prolonged development duration and less reproduction of P. marginatus than in susceptible cassavas. The insectresistant cassavas have a better capability to response to P. marginatus damage than susceptible cassavas. This study provided an example at the molecular, enzymatic activity and metabolite levels to analyse the physiological and biochemical occurrences of cassava and P. marginatus, which will contribute to a better understanding of the mechanism of plant resistance to insect herbivores. Our study can be used for the identification and breeding of mealybug-resistant cassava cultivars and help in the development of effective strategies for controlling P. marginatus populations.

Author contributions
QC conceived and designed this project and revised this manuscript. XQL and XL performed the experiments and wrote the manuscript. YL, CLW, XLX, QC, YW, XWY, YQ and JS contributed to sample collection and helped with data analysis. All authors contributed to the article and approved the submitted version.