Introduction
Cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a primary and important pest of cotton, bean, chickpea, corn, tobacco, tomato, pigeonpea, and other high-value agricultural crops in Iran and other countries worldwide (Fitt, Reference Fitt1989; Liu et al., Reference Liu, Li, Gong and Wu2004; Talekar et al., Reference Talekar, Opena and Hanson2006; Hemati et al., Reference Hemati, Naseri, Nouri-Ganbalani, Rafiee-Dastjerdi and Golizadeh2012a; Fallahnejad-Mojarrad et al., Reference Fallahnejad-Mojarrad, Goldasteh, Rafiei-Karahroodi and Vafaei Shoushtari2017; Atashi et al., Reference Atashi, Shishehbor, Seraj, Rasekh, Hemmati and Riddick2021). The use of synthetic chemical insecticides for the management of H. armigera has resulted in the development of insecticide resistance in the pest (Ismail, Reference Ismail2020). On the other hand, the use of pesticides in agriculture may impose a serious negative impact on the environment (Radwan et al., Reference Radwan, Youssef, Hashem and Shalaby2019; Hemmati et al., Reference Hemmati, Shishehbor and Stelinski2022). Due to the emergence of resistant insects and environmental risks of conventional insecticides, it is necessary to employ alternative pest management approaches that are more cost-effective, and sustainable (Korrat et al., Reference Korrat, Abdelmonem, Helalia and Khalifa2012).
Recent study efforts are focused on the use of resistant host plants, which can reduce the need for insecticides and improve the profitability, efficacy, and environmental acceptability of crop production (Kennedy et al., Reference Kennedy, Gould, Deponti and Stinner1987; Toews et al., Reference Toews, Cuperus and Phillips2000; Hemmati et al., Reference Hemmati, Takalloo, Taghdir, Mehrabadi, Balalaei, Moharramipour and Sajedi2021a). Moreover, plant resistance can be an economically beneficial, effective approach in integrated pest management and easy to use (Ofuya and Credlandt, Reference Ofuya and Credlandt1995; La Rossa et al., Reference La Rossa, Vasicek and Lopez2013; Shishehbor and Hemmati, Reference Shishehbor and Hemmati2022).
Feeding indices help evaluate the performance of pests in response to nutrition on various plant cultivars (Lazarevic and Peric-Mataruga, Reference Lazarevic and Peric-Mataruga2003; Hemati et al., Reference Hemati, Naseri, Nouri-Ganbalani, Rafiee-Dastjerdi and Golizadeh2012b). In addition to nutritional indices studies, understanding the insects' digestive physiology is also important when developing environmentally-safe strategies of control, such as using transgenic plants and digestive enzyme inhibitors (Jongsma and Bolter, Reference Jongsma and Bolter1997; Babamir-Satehi et al., Reference Babamir-Satehi, Habibpour, Aghdam and Hemmati2022). Evaluating the digestive function of insects on various host plants is a suitable tool for determining the relationship between host plants and herbivores that could improve the integrated pest management (IPM) strategies (Kotkar et al., Reference Kotkar, Sarate, Tamhane, Gupta and Giri2009; Hemati et al., Reference Hemati, Naseri, Nouri-Ganbalani, Rafiee-Dastjerdi and Golizadeh2012a; Zamani Fard et al., Reference Zamani Fard, Hemmati, Shishehbor and Stelinski2022). Furthermore, the standardized insect-growth index (SII) and the index of plant quality (IPQ) are used to evaluate the host plant quality for insect pests (Shishehbor and Hemmati, Reference Shishehbor and Hemmati2022).
Based on available studies, the level of infection with H. armigera varies considerably from cultivar to cultivar on host plants. Legumes are one of the most important plant sources of high levels of carbohydrates, calcium, iron, and especially protein and the second largest source of human food after cereals that widely cultivated as one of the most nutritious foods throughout world (Kotkar et al., Reference Kotkar, Sarate, Tamhane, Gupta and Giri2009; Sanon et al., Reference Sanon, Ba, Binso-Dabire and Pittendrigh2010). Although different researchers have studied the effects of host plants on feeding efficiency and digestive enzyme activity of H. armigera, there is no information about the effect of various broad bean cultivars on physiological characteristics and growth indices of this pest. Previous studies have been conducted on the effects of various cultivars of soybean on nutritional indices of cotton bollworm (Naseri et al., Reference Naseri, Fathipour, Moharramipour and Hosseininaveh2010a; Soleimannejad et al., Reference Soleimannejad, Fathipour, Moharramipour and Zalucki2010). Previously, Hemati et al. (Reference Hemmati, Takalloo, Taghdir, Mehrabadi, Balalaei, Moharramipour and Sajedi2012a, Reference Hemmati, Karam Kiani, Serrão and Jitonnom2021b) investigated the digestive proteolytic and amylolytic activities, feeding responses, and growth indices of this pest on various legumes including common bean, chickpea, tomato, white and red kidney bean, potato, and cowpea and indicated that tomato was a relatively unsuitable host plant for nutrition of larvae cotton bollworm.
Baghery et al. (Reference Baghery, Fathipour and Naseri2013) demonstrated the growth rate and nutritional performance of H. armigera on seeds of five host plants, including chickpea, cowpea, navy bean, corn, and soybean. They found that the cowpea and corn were more and less nutritious than the other hosts, respectively. Furthermore, Kouhi et al. (Reference Kouhi, Naseri and Golizadeh2014) studied nutritional indices of H. armigera on different tomato cultivars, and their results showed that the Rio grande UG cultivar is an unsuitable host for the cotton bollworm. Rahimi Namin et al. (Reference Rahimi Namin, Naseri and Razmjou2014) evaluated larval nutritional indices and activities of digestive enzymes in response to bean cultivars. They reported that red kidney bean Sayyad was the unsuitable host for feeding H. armigera. Furthermore, Naseri and Arghand (Reference Naseri and Arghand2015) determined the effect of an artificial diet containing seeds of five corn hybrids on the nutritional performance of this pest and screened the higher feeding performance of the larvae on SC260 and SC704 and lower on SC500 and SC700 hybrids. Fallahnejad-Mojarrad et al. (Reference Fallahnejad-Mojarrad, Goldasteh, Rafiei-Karahroodi and Vafaei Shoushtari2017), and Fathipour et al. (Reference Fathipour, Chegeni and Moharramipour2018) expressed that various cultivar plants significantly affected on growth and nutrition of H. armigera.
No published data were available in the correlation between tested parameters of cotton bollworm with phytochemical metabolites of legumes based on artificial diets. Therefore, the objectives of the present study were as follows: (A) feeding responses and digestive enzyme activities of H. armigera on an artificial diet based on different legume cultivars; (B) the investigation of some phytochemical metabolites in the tested legume seeds; and (C) analysis of the possible relationship between feeding properties and digestive function of H. armigera with biochemical compounds of various legume seeds. The outcomes of this study help identify plant inhibitors produced by legume cultivars that can be cloned into different host plants to confer protection against H. armigera.
Materials and methods
Legume seed source
Seeds of different legumes, including broad bean (Vicia faba L.; Shadan, Feyz, Saraziri, Barekat, Mahta cultivars), common bean (Phaseolus vulgaris L.; Khomein cultivar), white and red kidney bean (P. vulgaris; Goli and Dehghan cultivars), and cowpea (Vigna sinensis L.; Arabi and Mashhad cultivars) were obtained from the Agricultural Research Center of Safiabad, Dezful, Iran. Legumes were powdered separately and were placed in paper bags, kept in refrigerators at 4°C for larvae feeding. These legume seeds are commonly grown in Iran and used as food.
Cotton bollworm rearing
H. armigera larvae were collected from the tomato field in the Behbahan region (Khuzestan province) in the southwest of Iran in October 2020. The larvae of cotton bollworm were reared on an artificial diet based on the seed powder of each legume under controlled conditions: 25 ± 1°C, 60 ± 5% RH, and a photoperiod of 16:8 (L:D) hours. The artificial diet contained: powdered legume seed (125 g), wheat germ (15 g), yeast (17.5 g), sorbic acid (0.55 g), ascorbic acid (1.75 g), sunflower oil (2.5 ml), agar (7 g), methyl-p-hydroxy benzoate (1.1 g), formaldehyde 37% (1.25 g), and distilled water (325 ml) (Shorey and Hale, Reference Shorey and Hale1965). To prevent cannibalism, third instar larvae of cotton bollworm were transferred to individual Petri dishes and kept until pupation (Twine, Reference Twine1971). The adults of cotton bollworm were fed a honey solution (10%). Before starting the experiments, larvae were reared on an artificial diet based on each legume seed powder for two generations. Then the third-generation colony was used for the feeding indices and digestive enzymes experiments.
Nutritional performances of cotton bollworm
After mating, females of H. armigera that laid eggs were removed, and eggs were kept in Petri dishes with 8 cm diameter and 1 cm depth (40 eggs per cultivar). The first and second instar larvae of H. armigera were reared together in plastic dishes of 25 cm in height and 15 cm in width until the emergence of the third instar larvae. Then, the third instar larvae were separately reared in Petri dishes to prevent any cannibalistic behavior. From the third to fifth instar stage, the larval weight was measured before and after feeding on powdered artificial diets containing various legume until pre-pupation (25 replicates for each cultivar). The diets were replaced with new ones every 24 h. After daily feeding, the larval weight, the amount of the remaining leaves, and the feces produced by larvae were documented. To determine the percentage of dry weight of H. armigera food and larvae, 20 samples were weighed for each legume, dried in an oven at 60°C for 48 h and then weighed again. Nutritional performances of H. armigera larvae were calculated by Waldbauer (Reference Waldbauer1968) formulae:
where A = average of larval dry weight over time (mg), E = dry weight of the food consumed (mg), F = dry weight of feces produced, P = dry weight gain of larvae (mg), T = the feeding duration (day), and W 0 = primary weight of larvae (mg).
Moreover, the pupal weight of H. armigera was measured after 24 h of pupation on each cultivar. The SII was determined by Itoyama et al. (Reference Itoyama, Kawahira, Murata and Tojo1999) formula:
Furthermore, IPQ was determined using Koricheva and Haukioja (Reference Koricheva and Haukioja1992) formula:
Digestive enzyme assay
The fifth instar larvae of H. armigera reared on each legume seed were decomposed in pre-cooled distilled water under a stereomicroscope. The midguts of 20 larvae of H. armigera were homogenized on ice and prepared as described by Hosseininaveh et al. (Reference Hosseininaveh, Bandani, Azmayeshfard, Hosseinkhani and Kazzazi2007). Homogenates were centrifuged at 15,000g at 4°C for 10 min. The supernatants were collected and frozen (−20°C) for enzymatic assays. All assays were done in three replicates (per each legume cultivar) with a blank lacking midgut extract.
Amylase activity of cotton bollworm
Amylase activity of cotton bollworm larvae fed with different legumes was evaluated by the dinitrosalicylic acid (DNSA) method, with 1% starch as a substrate in the universal buffer system (10 mM succinate-glycine-2, morpholinoethan sulfunic acid) at pH 9. The mixtures containing midgut extracts and 1% starch were incubated at 37°C for 30 min. The enzymatic reaction was stopped by adding 50 μl of DNSA reagent and heating in boiling water for 15 min. The adsorption of mixture was read at 540 nm after cooling on ice (Bernfeld, Reference Bernfeld1955). The amount of maltose released during the α-amylase assays were estimated using the standard curve generated by using known amounts of maltose (2, 1, 0.5, 0.25, and 0.125 mg ml−1).
Protease activity of cotton bollworm
Proteolytic activity was examined using azocasein (1.5%) as substrate in the universal buffer system (50 mM sodium phosphate-borate) at pH 12, and reading the absorbance at 440 nm. The reaction mixture containing 50 μl of the midgut extract and 80 μl of the substrate in 50 mM universal buffer was incubated at 37°C for 50 min. Proteolysis was stopped by adding 100 μl of 30% trichloroacetic acid, followed by cooling at 4°C for 30 min and centrifugation at 14,000 ×g for 10 min. An equal volume of 2 M NaOH was added to the supernatant, and the absorbance was measured at 440 nm (Elpidina et al., Reference Elpidina, Vinokurov, Gromenko, Rudenshaya, Dunaevsky and Zhuzhikov2001). Furthermore, protein concentrations were estimated using the Bradford (Reference Bradford1976) protein assay. The standard curve was constructed using known amounts of bovine serum albumin (BSA) (2, 1.5, 1, 0.5, 0.25, 0.125, and 0.063 mg ml−1).
Biochemical properties of legume seeds
Biochemical characteristics of seeds of different legume cultivars, including protein, starch, total phenolic, and flavonoid amounts, were investigated to detect the relationship between biochemical compositions with feeding responses and enzyme activities of H. armigera. Biochemical assays were performed in three replicates for each cultivar, and distilled water was used as control. The powdered legumes were used to measure all phytochemicals of the tested cultivars.
The Bradford procedure was utilized to evaluate the protein content of the seeds. Briefly, 200 mg of the powdered seeds of each cultivar was homogenized in 10 ml of distilled water, and then 100 μl of the homogenate was mixed to 3 ml of Bradford reagent. The absorption was measured at 595 nm using BSA as a standard (Bradford, Reference Bradford1976).
The Bernfeld method (Reference Bernfeld1955) was utilized to estimate the starch content of legumes. The powdered legume seeds (0.2 g) were homogenized in 35 ml distilled water and heated to boiling. Then, 100 μl of each sample was mixed with 2.5 ml of iodine reagent (0.2% KI and 0.02% I2). The absorbance was recorded at 580 nm.
Furthermore, to measure the total phenol content, the Folin–Ciocalteau reagent was used (Sonald and Laima, Reference Sonald and Laima2001). Briefly, the powdered seeds were homogenized in methanol. After centrifugation, the supernatants were transferred to 1.5 ml Folin–Ciocalteau reagent 10% and sodium carbonate solution 7% was added to the mixture and incubated for 30 min at dark conditions. Standards and samples were measured with a spectrophotometer (S2100SUV, UNICO, USA) at 725 nm wavelength. Gallic acid was expressed quantified as the standard.
The method described by Jia et al. (Reference Jia, Tang and Wu1999) was used to determine the flavonoids content in the seeds. Briefly, 2 g of powdered seeds was homogenized in acidified ethanol (1 acid acetic:100 ethanol) and centrifuged at 13,000 ×g for 12 min. The homogenates were filtered with Whatman paper, and heated at 80°C in boiling water for 12 min. After cooling the filtrates, the absorbance was read at 430 nm, and the flavonoid content of seeds was calculated using a standard curve made by known amounts of catechin.
Data analysis
Normality of data was checked using the Kolmogorov–Smirnov test by the statistical software SPSS v. 22.0. Furthermore, the result of each cultivar experiment was analyzed by one-way analysis of variance. Statistical differences of means were assessed with the Tukey test at 5% significance level. Dendrogram of various legumes was performed based on all parameters tested for H. armigera by Ward's method. Pearson's correlation analysis was investigated to analyze the relationship between nutritional responses and enzyme activity of H. armigera with biochemical traits of legume cultivars (SPSS Inc., 2007).
Results
Feeding responses of cotton bollworm
Nutritional performance of third larval instar of H. armigera reared on the different legume cultivars is indicated in table 1. The highest value of consumption index (CI) was observed on Mahta (3.74) and Mashhad (3.65) cultivars. Furthermore, the lowest value of CI was on Shadan cultivar (2.43) (F = 7.42; df = 9, 240; P < 0.01). The highest approximate digestibility (AD) value of the third instar larvae was on Feyz cultivar (63.35%) and the lowest was on Arabi cultivar (43.11%) (F = 9.07; df = 9, 240; P < 0.01). The larvae reared on Dehghan cultivar had the highest efficiency of conversion of ingested food (ECI, 33.01%) (F = 60.92; df = 9, 240; P < 0.01), efficiency of conversion of digested food (ECD, 74.67%) (F = 35.03; df = 9, 240; P < 0.01), and relative growth rate (RGR, 0.347) (F = 100.14; df = 9, 240; P < 0.01) values. However, the lowest values of ECI (10.30%), ECD (20.45%), and RGR (0.083) indices were found on Khomein cultivar compared with the other hosts. Among the various host cultivars, the highest value of relative consumption rate (RCR) was observed on Arabi cultivar (1.299), and the lowest value was obtained on broad bean Shadan cultivar (0.690) (F = 17.95; df = 9, 240; P < 0.01) (table 1).
Table 1. Nutritional indices of the third instar larvae of Helicoverpa armigera fed on artificial diets based on the different legume seeds
CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RCR, relative consumption rate; RGR, relative growth rate.
The means followed by different letters in the same column are significantly different (Tukey, P < 0.01).
The nutritional indices of the fourth instar larvae of the cotton bollworm were significantly different on the various legume seeds tested. The larvae fed with Khomein cultivar (3.26) had the highest CI value, and the lowest value was observed when the larvae were fed with both Shadan (2.24) and Barekat (2.26) cultivars (F = 7.14; df = 9, 240; P < 0.01). The highest values of ECI index were recorded on both Goli (20.68%) and Dehghan (16.62%) cultivars, however, the lowest one was observed on Mahta (8.16%) and Khomein (8.40%) cultivars (F = 31.96; df = 9, 240; P < 0.01). The maximum values of ECD were found on both Dehghan (72.46%) and Goli (71.20%) cultivars and the minimum value was obtained on the Khomein cultivar (22.52%) (F = 40.01; df = 9, 240; P < 0.01). The cotton bollworm larvae reared on Dehghan (0.266) and Goli (0.252) cultivars had the highest values of RGR index (F = 81.688; df = 9, 240; P < 0.01). Among the various legume species, Dehghan cultivar had the highest value (1.41) of RCR (F = 24.28; df = 9, 240; P < 0.01) (table 2).
Table 2. Nutritional indices of the fourth instar larvae of Helicoverpa armigera fed on artificial diets based on the different legume seeds
CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RCR, relative consumption rate; RGR, relative growth rate.
The means followed by different letters in the same column are significantly different (Tukey, P < 0.01).
The highest CI of the fifth instar larvae was observed on the Khomein cultivar (1.70), whereas the lowest CI index (1.00) was found on the Barekat cultivar (F = 6.457; df = 9, 240; P < 0.01). The highest AD index was found in the cotton bollworm fed on the Feyz cultivar (38.28%), and the lowest one was observed on the Arabi cultivar (24.68%) (F = 3.649; df = 9, 240; P < 0.01). The larvae fed with the Dehghan cultivar showed the highest ECI (21.56%) and ECD (69.97%) values, while the lowest ECI index was achieved on Mahta cultivar (5.78%) and the minimum values of ECD were found on the Mahta (23.14%) and Khomein (24.88%) cultivars (ECI, F = 35.22; df = 9, 240; P < 0.01; ECD, F = 21.36; df = 9, 236; P < 0.01). The fifth instar larvae of H. armigera reared on Dehghan (0.165) and Goli (0.155) cultivars obtained the highest values of RGR. In contrast, the lowest RGR value was recorded when the larvae were fed with Mahta cultivar (0.037) (F = 39.71; df = 9, 240; P < 0.01). The maximum value of RCR index was recorded on the Goli (0.961) and Mashhad (0.956) cultivars and the lowest value was recorded on the Barekat cultivar (0.445) (F = 17.35; df = 9, 240; P < 0.01) (table 3).
Table 3. Nutritional indices of the fifth instar larvae of Helicoverpa armigera fed on artificial diets based on the different legume seeds
CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RCR, relative consumption rate; RGR, relative growth rate.
The means followed by different letters in the same column are significantly different (Tukey, P < 0.01).
There were significant differences among all nutritional response parameters of the total larvae (third to fifth instars) of cotton bollworm on various legume seeds tested. The larvae fed on the Khomein (2.46) and Barekat (1.72) cultivars had the highest and lowest CI index (F = 9.29; df = 9, 240; P < 0.01). The highest value of AD index was obtained on the all Feyz (45.50%), Barekat (42.83%) and Shadan (42.42%) cultivars, while the lowest one was documented on Arabi cultivar (31.01%) (F = 7.441; df = 9, 240; P < 0.01). The highest ECI index was achieved by rearing larvae on Dehghan cultivar (22.34%) and the lowest value was found on both Khomein (8.30%) and Mahta (8.48%) cultivars (F = 121.43; df = 9, 240; P < 0.01). Total instar larvae fed on Dehghan (70.71%) and Goli (62.18%) cultivars found the highest ECD value, while the lowest ECD index was documented on both Mahta (22.14%) and Khomein (22.53%) cultivars (F = 71.01; df = 9, 240; P < 0.01). The larvae of cotton bollworm fed with Dehghan (0.201 mg mg−1 day−1) and Goli (0.192) cultivars showed the highest values of RGR index. Furthermore, the lowest RGR value was achieved when the larvae were fed with Khomein cultivar (0.059) (F = 139.59; df = 9, 240; P < 0.01). The maximum value of RCR index was obtained on the Mashhad (1.02), Goli (0.95), and Arabi (0.93) cultivars and the minimum value was recorded on Barekat cultivar (0.602) (F = 31.28; df = 9, 240; P < 0.01) (table 4).
Table 4. Nutritional indices of whole instar larvae of Helicoverpa armigera fed on artificial diets based on the different legume seeds
CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RCR, relative consumption rate; RGR, relative growth rate.
The means followed by different letters in the same column are significantly different (Tukey, P < 0.01).
The results in fig. 1 show that the highest whole larval instars weights (75.01) (F = 27.92; df = 9, 240; P < 0.01) and feces produced values (97.90 mg) (F = 7.320; df = 9, 240; P < 0.01) were achieved on Dehghan cultivar, and the lowest larval weights were achieved in larvae fed on Khomein (52.16 mg) and Mahta (52.38 mg) cultivars (fig. 1a, c). The highest value of food consumed was detected on the Mashhad cultivar (153.21 mg), and the lowest was also recorded on the Mahta cultivar (113.91 mg) (F = 7.320; df = 9, 240; P < 0.01) (fig. 1b). The larvae of cotton bollworm fed on the Dehghan cultivar (31.58 mg) observed the maximum value of larval gain weight (F = 109.66; df = 9, 240; P < 0.01) (fig. 2d).
Figure 1. (a) Mean larval weight, (b) food consumed, (c) feces produced, and (d) larval gain weight of Helicoverpa armigera fed on artificial diets based on the different legume seeds.
Figure 2. Index of plant quality (IPQ) and standardized insect-growth index (SII) of Helicoverpa armigera fed on artificial diets based on the different legume seeds.
IPQ and SII indexes of cotton bollworm
There were significant differences in the SII and IPQ values of H. armigera fed on different host plant cultivars. The highest SII (F = 75.44; df = 9, 240; P < 0.01) and IPQ (F = 25.66; df = 9, 240; P < 0.01) indexes were obtained when the larvae were reared on the Dehghan cultivar (SII: 3.014; IPQ: 0.676), whereas the larvae reared on the Khomein cultivar had the lowest values (SII: 1.50; IPQ: 0.44) (fig. 2).
Enzyme activity of cotton bollworm
The activities of digestive proteases and amylases in fifth instar larval reared on artificial diets based on the different legume seeds are presented in fig. 3. The maximum and minimum proteolytic activity of the fifth instars were obtained in the larvae reared on Dehghan (1.65 U mg−1) and Barekat (0.498 U mg−1) cultivars (F = 6.86; df = 9, 20; P < 0.01) (fig. 3a). Moreover, the larvae fed on Mashhad cultivar showed the highest levels of amylolytic activity (4.98 mU mg−1), in contrast, no significant difference in amylolytic activity of fifth instar larvae value was recorded among larvae feeding on the other cultivars (F = 10.54; df = 9, 20; P < 0.01) (fig. 3b).
Figure 3. General proteolytic (a) and amylolytic (b) activity of midgut extracts from Helicoverpa armigera fed on artificial diets based on the different legume seeds.
Cluster analysis
The dendrogram-based feeding indices and growth parameters with enzymatic activity of H. armigera on different legume cultivars are presented in fig. 4. Two clusters A and B are apparent in the dendrogram. Sub-cluster A1 includes Feyz, Shadan, and Barekat cultivars, and sub-cluster A2 includes Mahta and Khomein cultivars. Sub-cluster B1 consisted of Saraziri and Mashhad cultivars and Dehghan, Goli, and Arabi cultivars were in sub-cluster B2 (fig. 4).
Figure 4. Dendrogram of different cultivars based on nutritional and growth indices and enzymatic activity of Helicoverpa armigera fed on artificial diets based on the different legume seeds (Ward's method).
Biochemical characteristics of various legume seeds
The primary and secondary metabolites of the tested legume cultivars are given in table 5. There were significant differences in the phytochemical metabolites of various seed cultivars. The highest protein content was measured in the Dehghan cultivar (0.342 mg ml−1) (F = 31.23; df = 9, 20; P < 0.01). In contrast, the lowest amount of starch seeds was achieved in the Mahta cultivar (1.81 mg ml−1) (F = 6.58; df = 9, 20; P < 0.01). The highest total phenolic content was detected in the Mahta (11.98 mg ml−1), Feyz (12.46 mg ml−1), and Shadan (11.97 mg ml−1) cultivars, and the lowest content was observed in the Dehghan cultivar (9.21 mg ml−1) (F = 10.79; df = 9, 20; P < 0.01). Furthermore, the highest amount of flavonoids was obtained for the Saraziri (111.80 mg ml−1), Feyz (111.93 mg ml−1), and Shadan (112.20 mg ml−1) cultivars, whereas the lowest amount was measured for Arabi (93.60 mg ml−1) and Mashhad (94.13 mg ml−1) cultivars (F = 60.39; df = 9, 20; P < 0.01).
Table 5. Biochemical characteristics (mean ± SE) (mg ml−1) of tested different legume seeds
The means followed by different letters in the same column are significantly different (Tukey, P < 0.01).
Correlation analysis
The analysis of correlation coefficients of nutritional performances and growth characteristics of H. armigera with their enzymatic activity when fed on different legume is given in table 6. Significant correlations were detected between feeding and growth of cotton bollworm and the amylolytic and proteolytic enzymes activity on various legume cultivars. The food consumed, larval gain, feces produced, RCR, and RGR parameters of H. armigera showed significant negative correlations with amylolytic and proteolytic enzymes activity (P < 0.01). In contrast, the digestive enzymes of larvae positively correlated with the tested growth characteristics of H. armigera, including SII and IPQ indexes (P < 0.01). There was no significant correlation among larval weight, AD, ECI, and ECD parameters of cotton bollworm with the amylolytic enzyme activity of larvae. Likewise, the CI and AD variables of H. armigera were not significantly correlated with amylolytic activity (P > 0.05). Furthermore, a significant negative correlation was observed between larval weight, ECI, ECD, and proteolytic enzyme activity of H. armigera (P < 0.01) (table 6).
Table 6. Correlation coefficients (r) of nutritional characteristics of Helicoverpa armigera with their digestive enzymes fed on various legume cultivars
SII, standardized insect-growth index; IPQ, index of plant quality; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RCR, relative consumption rate; RGR, relative growth rate. The number in parenthesis is P value.
Correlations were evaluated based on Pearson's correlation test (P < 0.01).
In addition, the analysis of correlation coefficients between nutritional indices and enzyme activity of H. armigera with biochemical traits of the tested cultivars is illustrated in table 7. SII index, amylolytic, and proteolytic activities of H. armigera showed a significant high positive correlation with protein content. In contrast, the food consumed, larval gain, and ECI, ECD, RCR, and RGR indices of larvae negatively correlated with protein content of legume cultivars (P < 0.01). There were significant negative or positive correlations between all nutritional and physiological traits of H. armigera with the starch amounts. The food consumed, larval gain, ECD, and RGR variables of larvae were significantly positively correlated with total phenolic content of the legume seeds. Furthermore, significant negative correlation was detected between total phenolic content of various legumes with SII parameter and amylolytic and proteolytic enzymes activity of larvae (P < 0.01). SII and IPQ variables and enzymes activity of H. armigera showed negative correlations with flavonoids content; conversely, food consumed, larval gain, ECI, ECD, RCR, and RGR indices of larvae were positively correlated with this biochemical trait of the studied legumes (table 7).
Table 7. Correlation coefficients (r) of nutritional and physiological characteristics of Helicoverpa armigera with biochemical traits of various legume cultivars
SII, standardized insect-growth index; IPQ, index of plant quality; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RCR, relative consumption rate; RGR, relative growth rate. The number in parenthesis is P value.
Correlations were evaluated based on Pearson's correlation test (P < 0.01).
Discussion
The growth rate of H. armigera can be affected by quantity and quality of food consumed. Our study showed that feeding properties and proteolytic and amylolytic activities of H. armigera were significantly affected by artificial diets based on the different legume seeds. These results are consistent with the previous studies related to H. armigera fed on various plants (Soleimannejad et al., Reference Soleimannejad, Fathipour, Moharramipour and Zalucki2010; Hemati et al., Reference Hemati, Naseri, Nouri-Ganbalani, Rafiee-Dastjerdi and Golizadeh2012b; Baghery et al., Reference Baghery, Fathipour and Naseri2013; Rahimi Namin et al., Reference Rahimi Namin, Naseri and Razmjou2014; Naseri and Arghand, Reference Naseri and Arghand2015; Fallahnejad-Mojarrad et al., Reference Fallahnejad-Mojarrad, Goldasteh, Rafiei-Karahroodi and Vafaei Shoushtari2017).
The quality of food may affect the life table, feeding performance, and nutritional physiology of insects, and these traits can indicate the degree of plant resistance. Furthermore, the importance of nutrient quality or phytochemical metabolites and physical characteristics of host plants, in insect–plant interactions, is demonstrated (Kennedy et al., Reference Kennedy, Gould, Deponti and Stinner1987; Gvozdenac et al., Reference Gvozdenac, Prvulovi, Radovanovi, Ovuka, Mikli, Acanski, Tanasković and Vukajlović2018; Shishehbor and Hemmati, Reference Shishehbor and Hemmati2022).
To the best of our knowledge, no findings exist on the nutritional physiology studies of this pest on various broad bean cultivars. The present research confirmed the findings of previous studies by others that low host quality, such as the presence of inhibitors, reduces feed and growth of H. armigera (Naseri et al., Reference Naseri, Fathipour, Moharamipour, Hosseininaveh and Gatehouse2010b; Hemati et al., Reference Hemati, Naseri, Nouri-Ganbalani, Rafiee-Dastjerdi and Golizadeh2012b; Kouhi et al., Reference Kouhi, Naseri and Golizadeh2014; Fathipour et al., Reference Fathipour, Chegeni and Moharramipour2018). The study results showed that H. armigera fed on the artificial diets based on the various legume cultivars could develop and feed at the different rates. Among different tested legumes, the highest CI index value of fourth, fifth, and whole instar larvae of cotton bollworm was on Khomein cultivar. Furthermore, the rate of food intake relative to average larval weight was highest during the feeding period, while the larvae fed with this cultivar.
The findings indicated that the fifth instar larvae reared on the Feyz cultivar had the highest AD value. It seems that the high AD index could not compensate for the low ECD index, which consequently resulted in growth retardation. Another reason may be the increased instar duration, in which large amounts of ingested food must be allotted to maintain metabolism (Scriber and Slansky, Reference Scriber and Slansky1981).
The highest ECI value for whole instar larvae on the artificial diets containing Dehghan cultivar indicated that the larvae were able to convert digested food to body mass and the weights of larvae improved on this cultivar. The ECI index measures an insect's ability to incorporate ingested food into growth (Lazarevic and Peric-Mataruga, Reference Lazarevic and Peric-Mataruga2003). Variation in ECD value can be related to differences in the physiological and biochemical characteristics of the ingested food (Nathan et al., Reference Nathan, Chung and Murugan2005). The lowest ECD value in the larvae of H. armigera was expressed on the Khomein and Mahta cultivars, suggesting the higher metabolic cost affecting catabolism, and, consequently, excretion (Scriber and Slansky, Reference Scriber and Slansky1981).
Furthermore, the feeding duration is an effective factor in the RCR and RGR values (Hemati et al., Reference Hemati, Naseri, Nouri-Ganbalani, Rafiee-Dastjerdi and Golizadeh2012b). Among various legume cultivars, the lowest RCR value of the fifth and whole larval instars of cotton bollworm was obtained in the Barekat cultivar. Probably, the Barekat cultivar has low nutritive content for larvae, and consequently, a longer immature period was needed to complete growth. Conversely, the highest RGR value of whole instar larvae of H. armigera on artificial diets containing Goli and Dehghan seeds indicates that the rate of increase in larval weight was the highest on these legumes. It is reported that the nutritional responses of an insect are positively correlated to body biomass (Nathan et al., Reference Nathan, Chung and Murugan2005). The lowest RGR value of whole instar larvae of cotton bollworm on Khomein cultivar may be due to decreased food consumption when the ingested food must be allocated to maintenance metabolism. The difference in the RGR index on various legume cultivars might be attributed to differences in primary and secondary plant metabolites among the legumes.
It has been shown that variations in allelochemical and biochemical traits and macronutrients among host plants could affect pest's performance (Tsai and Wang, Reference Tsai and Wang2001; Chen et al., Reference Chen, Ni and Buntin2009). The findings revealed that various legume species affect the nutritional indices of H. armigera, such as Dehghan and Goli cultivars as the suitable and Khomein and Mahta cultivars as the unsuitable host for feeding and developing of this pest.
In the present research, the lowest SII value in the larvae fed on Khomein cultivar may be related to a longer larval period or lower pupal weight on this cultivar. Additionally, the lowest value of IPQ on the Khomein cultivar can be correlated with a lower pupal weight and higher frass dry weight on this cultivar. The high values of feces production and food consumption on the Khomein cultivar might be related to the higher contents of secondary metabolites in this cultivar compared to the other cultivars (Shishehbor and Hemmati, Reference Shishehbor and Hemmati2022).
Previous studies have shown that insects, especially polyphagous pests, rapidly modify their digestive enzyme profiles in response to ingested plant protease inhibitors by down and up adjustment of gut proteases (Harsulkar et al., Reference Harsulkar, Giri, Patankar, Gupta, Sainani, Ranjekar and Deshpande1999; Hemmati et al., Reference Hemmati, Takalloo, Taghdir, Mehrabadi, Balalaei, Moharramipour and Sajedi2021a). In this study, protease and amylase as main digestive enzymes in midgut of H. armigera were considered. Digestive proteases play two major functions in insect physiology including inactivating protein toxins consumed as a consequence of feeding and breaking down proteins into amino acids essential for growth and development (Nation, Reference Nation2002). The number of proteins in legume seeds may affect the major insect digestive enzymes such as proteases (Biggs and McGregor, Reference Biggs and McGregor1996). Inactivation of digestive enzymes by inhibitors leads to blocking gut proteases and other digestive enzymes such as amylases, leading to poor nutrient utilization, growth retardation, and death because of starvation (Hemmati et al., Reference Hemmati, Karam Kiani, Serrão and Jitonnom2021b).
The enzymatic activity of H. armigera was affected with food eaten, that reported by other researchers (Hemati et al., Reference Hemati, Naseri, Nouri-Ganbalani, Rafiee-Dastjerdi and Golizadeh2012a; Rahimi Namin et al., Reference Rahimi Namin, Naseri and Razmjou2014; Fallahnejad-Mojarrad et al., Reference Fallahnejad-Mojarrad, Goldasteh, Rafiei-Karahroodi and Vafaei Shoushtari2017). The low gut amylolytic activity of cotton bollworm in all cultivars (except Mashhad legume) may be related to the presence of amylase inhibitors in these legume seeds. Moreover, the low gut proteolytic activity level in larvae fed on artificial diet containing Barekat cultivar might be correlated to the ingested protease inhibitors or the low protein content in this legume. The role of amylase and protease inhibitors on the level of enzymatic activity in H. armigera is known and should be demonstrated in future research.
In this study, cluster analysis showed that concerning growth and nutritional indices and enzymatic activity of H. armigera, various legume cultivars could be divided into four distinct classes. The grouping in each class might be due to a high level of physiological similarity of various legume cultivars. Cluster A1 included the Feyz, Shadan, and Barekat, and cluster B1 consisted of Saraziri and Mashhad cultivars as intermediate groups. Cluster A2 included Mahta and Khomein cultivars as relatively unsuitable group, which could be due to the lowest values of IPQ and the highest values of secondary metabolites in these cultivars. Cluster B2 consisted of Dehghan, Goli, and Arabi cultivars as relatively suitable group.
The present study suggests the existence of interactions between feeding parameters and activity of digestive enzymes of H. armigera with the biochemical metabolites of legume cultivars. Significant negative or positive correlations were found in correlation analysis between growth characteristics and digestive enzymes activity of cotton bollworm with primary and secondary metabolites of legume seeds. Correlation analysis showed that the growth and nutrition of H. armigera fed on various legume cultivars were influenced by the digestive enzyme activity of this pest. The rate of feeding in reared insects was the lowest in Mahta and Khomein cultivars, probably due to low nutritional level, biochemical properties, and high concentration of protein inhibitors, indicating that larvae feeding on these cultivars were less effective in converting ingested and digested food to biomass.
Correlation analysis revealed that amylolytic and proteolytic activities of H. armigera were significantly negatively correlated with the many nutritional and growth characteristics of H. armigera on different legume seeds. Furthermore, a positive correlation was observed between high amylolytic and proteolytic activities in the midgut with the high SII index of H. armigera larvae fed on Goli and Dehghan cultivars, suggesting that the quality and nutritional value of these legumes are suitable for the feeding of larvae. Variations in the protein contents of host plants might lead to differences in the proteolytic activity of H. armigera feeding on these plants.
In conclusion, the present results revealed significant differences in the nutritional performance and activities of major digestive enzymes of H. armigera larvae reared on diets containing different legume seeds. By combining the findings of the present research about the feeding responses and proteolytic and amylolytic activities of larvae fed on different legumes, it is highly probable that the Mahta and Khomein cultivars contain plant inhibitors that mediate antibiosis to insects reflected by the poor performance of H. armigera on these cultivars. Further research is required to identify the plant compound(s) responsible for delaying growth and development of H. armigera. Identification of these inhibitory compounds in host plants provides an interesting opportunity to generate transgenic pest-resistant plants as a sustainable pest management strategy. Moreover, a wider range of plants can be tested for selecting cultivars that are more resistant to H. armigera. A comprehensive biochemical and molecular analysis of midgut proteases and carbohydrases may shed light on the adaptive responses of larvae to feeding on different cultivars.
Acknowledgements
This research was financially supported by Shahid Chamran University of Ahvaz, Ahvaz, Iran (Grant No. SCU.AP99.39134), which is greatly appreciated.
Conflict of interest
None.
