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Evaluation of diets from various maize hybrids reveals potential tolerance traits against Spodoptera littoralis (Boisd) as measured by developmental and digestive performance

Published online by Cambridge University Press:  27 September 2024

Shirin Alekaram
Affiliation:
Department of Plant Protection, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran
Seyed Ali Hemmati*
Affiliation:
Department of Plant Protection, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran
Masumeh Ziaee
Affiliation:
Department of Plant Protection, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran
Lukasz L. Stelinski
Affiliation:
Department of Entomology and Nematology, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, 33850, USA
*
Corresponding author: Seyed Ali Hemmati; Email: [email protected]
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Abstract

Spodoptera littoralis (Boisd) (Lepidoptera: Noctuidae) is a highly polyphagous insect that significantly reduces agricultural production of several food staples. We evaluated performance of S. littoralis on several meridic diets based on various maize hybrids, including Oteel, Simon, Valbum, SC703, and SC704. Growth, feeding behaviours, and activity of digestive enzymes of S. littoralis were examined under laboratory conditions. In addition, selected biochemical characteristics of maize hybrid seeds were evaluated, including starch, protein, anthocyanin, as well as phenolic and flavonoid contents, to examine relationships between plant properties and digestive performance of S. littoralis. Performance of S. littoralis on maize hybrids, as measured by nutritional indices, was related to both proteolytic and amylolytic activities quantified using gut extracts. Larval S. littoralis reared on SC703 exhibited the highest efficiency of conversion of digested food, while the lowest was recorded in those fed on the Oteel hybrid. S. littoralis reared on SC703 and Oteel also exhibited the highest and lowest relative growth rates, respectively. The highest levels of proteolytic activity in S. littoralis were measured from larvae reared on the SC703 hybrid, while the lowest levels occurred on the Oteel and Valbum hybrids. Amylolytic activity was lowest in larvae reared on SC703 and Valbum hybrids and highest in larvae reared on the Oteel hybrid. Our results suggest that the SC703 hybrid was the most suitable host for S. littoralis, while the Oteel hybrid demonstrated the greatest level of tolerance against S. littoralis of those evaluated. We discuss the potential utility of maize hybrids exhibiting tolerance traits against this cosmopolitan pest with reference to cultivation of tolerant varieties and identification of specific tolerance traits.

Type
Research Paper
Copyright
Copyright © The Author(s), 2024. Published by Cambridge University Press

Introduction

Maize, Zea mays L., is a monocotyledonous annual crop cultivated globally given its many uses, high nutritional value, and adaptability to diverse climates (especially with the production of new hybrids). Maize ranks third in the world after wheat and rice in terms of cultivated area and total production (Khodabandeh, Reference Khodabandeh2003; Erenstein et al., Reference Erenstein, Jaleta, Sonder, Mottaleb and Prasanna2022). Maize yield potential per unit area is high allowing harvest of 15 to 20 tons of seeds per hectare at the commercial level. Maize has been called the king of grains due to its capacity for seed production (Scott and Emery, Reference Scott, Emery, Wrigley, Corke, Seetharaman and Faubion2016).

One of the important pests of maize is the Egyptian cotton leafworm, Spodoptera littoralis (Boisd) (Lepidoptera: Noctuidae), which significantly reduces maize production annually (Gouinguené et al., Reference Gouinguené, Alborn and Turlings2003; Khanjani, Reference Khanjani2006; El-latif, Reference El-latif2014; Zamani Fard et al., Reference Zamani Fard, Hemmati, Shishehbor and Stelinski2022). Also, S. littoralis is one of the most important pests of other high value crops such as cotton, beans, alfalfa, clover, sugar beet, and vegetables in southern Europe, Africa, and the Middle East (Hegazi and Schopf, Reference Hegazi and Schopf1984; Hosseini Mousavi et al., Reference Hosseini Mousavi, Hemmati and Rasekh2023). S. littoralis larvae damage vegetative and reproductive parts of agricultural crops, including cotton, tomato, maize, and beans (Sneh et al., Reference Sneh, Schuster and Broza1981; El-latif, Reference El-latif2014; Ismail, Reference Ismail2020). The larvae feed collectively on the parenchyma of young and old leaves, which become reticulated, and can be completely skeletonised under high population densities (Khanjani, Reference Khanjani2006; Lanzoni et al., Reference Lanzoni, Bazzocchi, Reggiori, Rama, Sannino, Maini and Burgio2012).

Typically, farmers use broad-spectrum insecticides to control S. littoralis (Ismail, Reference Ismail2020; Hosseini Mousavi et al., Reference Hosseini Mousavi, Hemmati and Rasekh2023). Insecticide overuse can cause harmful side effects, including evolution of resistance, destruction of natural enemies, reduced crop quality, and secondary pest outbreaks (Ongley, Reference Ongley and Ongley1996; Smagghe et al., Reference Smagghe, Carton, Wesemael, Ishaaya and Tirry1999; Gacemi et al., Reference Gacemi, Taibi, Abed, M'hammedi Bouzina, Bellague and Tarmoul2019). Therefore, alternative approaches for management of S. littoralis have been prioritised in many regions. At present, identification of tolerance traits to breed for pest resistant crops represents one environmentally sound approach to managing this pest that can also be economical for growers in developing nations (Panda and Khush, Reference Panda and Khush1995; Liu et al., Reference Liu, Li, Gong and Wu2004; Tsai and Wang, Reference Tsai and Wang2006; Hemmati et al., Reference Hemmati, Shishehbor and Stelinski2022). Also, incorporating resistant plant cultivars can supplement chemical and biological control methods in IPM programmes (Sharma and Ortiz, Reference Sharma and Ortiz2002; Lacey et al., Reference Lacey, Grzywacz, Shapiro-ilan, Frutos, Brownbridge and Goettel2015; Jafari et al., Reference Jafari, Hemmati and Habibpour2022, Reference Jafari, Habibpour, Hemmati and Stelinski2023).

The growth, development, and reproduction of insects strongly depend on the quality and quantity of their food (Scriber and Slansky, Reference Scriber and Slansky1981; Razmjou et al., Reference Razmjou, Naseri and Hemati2014). In response to insect herbivory, plants produce a series of biochemical metabolites, which may reduce digestibility as well as deterrents compounds, which can inhibit feeding (Smith and Clement, Reference Smith and Clement2012; Zamani Fard et al., Reference Zamani Fard, Hemmati, Shishehbor and Stelinski2022). For example, plant protease inhibitors (PIs) interfere with the activity of digestive function in insects, thereby reducing assimilation of diets and resulting in nutrient deficiency, deceased development, and increased larval mortality (Stevens et al., Reference Stevens, Dunse, Fox, Evans, Anderson and Soundararajan2012; Hemati et al., Reference Hemati, Naseri, Nouri-Ganbalani, Rafiee-Dastjerdi and Golizadeh2012a). Previous studies have indicated that seeds from cereals (Gramineae), such as maize, are a rich source of antifeedant compounds and PIs from a diverse group of feeding inhibitor families (Franco et al., Reference Franco, Rigden, Melo and Grossi-de-Sá2002; Gomes et al., Reference Gomes, Barbosa, Macedo, Pitanga, Moura, Oliveira, Moura, Queiroz, Macedo, Andrade, Vidal and Sales2005; Silva et al., Reference Silva, Casado-Filho, Corrêa, Farias, Bloch, de Sá MF, Mendes, Quirino, Noronha and Franco2007; El-latif, Reference El-latif2014). Furthermore, PI-producing plant cultivars show promise when incorporated as part of pest management programmes due to their tolerance traits (Gatehouse and Gatehouse, Reference Gatehouse and Gatehouse1998; Mehrabadi et al., Reference Mehrabadi, Franco, Bandani and Bandani2012).

Tolerance against S. littoralis has been investigated previously among various legume cultivars. For example, cowpea Mashhad exhibits considerable tolerance against this pest (Shishehbor and Hemmati, Reference Shishehbor and Hemmati2022). Zamani Fard et al. (Reference Zamani Fard, Hemmati, Shishehbor and Stelinski2022) also investigated the digestive enzyme activities and feeding efficiency of S. littoralis on different mung bean varieties and reported that the VC6371 variety was a comparatively vulnerable cultivar to this pest. Recently, Hosseini Mousavi et al. (Reference Hosseini Mousavi, Hemmati and Rasekh2023) investigated performance of S. littoralis on various leafy vegetables, and found that coriander was a suitable host, while purslane exhibited tolerance.

The objectives of this study were to quantify the performance of S. littoralis on meridic diets derived from various maize hybrids and to measure the response of major digestive enzyme groups in this insect after feeding on these potential hosts. Moreover, we measured various primary and secondary metabolites including protein, carbohydrate, and phenolic content as well as flavonoids and anthocyanins among seeds of these maize hybrids. Finally, we examined relationships between growth indices or digestive enzyme responses of S. littoralis and the primary and secondary metabolite levels found among the hybrids evaluated. Our results provide a basis for selection of available maize hybrids for cultivation based on their level of tolerance against S. littoralis. Incorporating plant tolerance into management programmes where S. littoralis causes significant damage should be a useful additional consideration, particularly in areas where economic means limit availability of chemical tools.

Material and methods

Maize seeds

Maize seeds from the hybrids Oteel, Simon, Valbum, SC703, and SC704, commonly cultivated for commercial purposes in Iran, were obtained from the Safiabad Dezful Agriculture and Natural Resources Research and Education Center, Dezful, Iran. The seeds were individually ground and stored at 4°C before use in the experiments. Meridic diets were separately prepared from seed powder of each plant hybrid (250 g) and with the addition of wheat germ (30 g) for supplemental protein and carbohydrate sources, sorbic acid (1.1 g) as an antimicrobial agent, ascorbic acid (3.5 g) as a vitamin source, sunflower oil (5 ml) as a lipid source, agar (14 g) as a moisturiser, methyl p-hydroxybenzoate (2.2 g), formaldehyde 37% (2.5 g), and distilled water (650 ml). The meridic diets were prepared based on the method described by Shorey and Hale (Reference Shorey and Hale1965) and were stored in the refrigerator for no longer than two weeks prior to use.

Insect rearing

The initial population of S. littoralis was collected from maize fields in Khuzestan province of southwestern Iran, and reared in a growth chamber at 25 ± 1°C, 60 ± 5% RH, and a of 16:8 h (L:D) photoperiod. The collected larvae were placed in plastic containers (diameter 9 cm, depth 1 cm) and reared on the various maize-based meridic diets. Adults that emerged were kept in plastic containers (15 cm diameter × 20 cm height) and provided wet cotton soaked in honey solution (10%). Prior to initiating experiments, larvae were reared on diets consisting of all five maize hybrids for two generations to create a homogenous cohort.

Food consumption and growth indices of Spodoptera littoralis

Forty S. littoralis eggs of the same age were selected from the main culture for subsequent rearing per maize hybrid diet. Until third instar larvae appeared, first and second instars were reared as groups of 40 larvae in 25 × 15 cm (height × width), plastic containers. After third instars emerged, 25 larvae were chosen at random for each maize hybrid diet and placed individually into 8 × 1 cm Petri dishes with diet to prevent cannibalism. Petri dish lids were modified by drilling 2 cm diameter holes in the lids that were securely covered with a net cloth to allow ventilation. Humidity was maintained by adding cotton soaked with deionised water in the rearing container. Recording of nutritional indices for S. littoralis was initiated after emergence of third instar larvae. Daily weight measurements were taken, including the weight of the larvae before and after eating, the weight of the frass produced, and the weight of the initial and leftover food. S. littoralis were also weighed at pre-pupal and pupal stages within 24 h of appearance. All weights were measured using a digital scale with an accuracy of 0.001 g. Moreover, the dry weights of the meridic diet samples, as well as larvae and their frass, were calculated by first weighing 25 samples per maize cultivar, then oven-drying the samples at 60°C for 48 h, and then re-weighing those samples again.

Nutritional performance of S. littoralis larvae on meridic diets based on various maize hybrids was estimated by calculating several indices, including approximate digestibility (AD), a consumption index (CI), the efficiency of conversion of digested food (ECD), the efficiency of conversion of ingested food (ECI), relative growth rate (RGR) and relative consumption rate (RCR), using the formulas by Waldbauer (Reference Waldbauer1968):

$$\eqalign{& {\rm CI} = [ {( {E/A} ) } ] ; \;{\rm AD} = [ {( {E-F} ) /E} ] ; \;{\rm ECI} = [ {( {P/E} ) \,\times \,100} ] ; \;\cr & {\rm ECD} = [ {( {P/E-F} ) \,\times \,100} ] ; \;{\rm RCR} = [ {( {E/W_0\,\times \,T} ) } ] ; \cr & {\rm and\ RGR} = [ {P/W_0\,\times \,T} ] .} $$

where A = average of larval dry weight over time (mg), E = dry weight of the food consumed (mg), F = dry weight of faeces produced, P = dry weight gain of larvae (mg), T = the feeding duration (day), and W 0 = primary weight of larvae (mg).

Moreover, to calculate the standardised insect-growth index (SII), pupal weight (P w) was divided by the larval duration (T) (Itoyama et al., Reference Itoyama, Kawahira, Murata and Tojo1999). In order to determine the index of plant quality (IPQ) for the maize hybrids investigated here, pupal weight was divided by the dry weight of insect frass (Koricheva and Haukioja, Reference Koricheva and Haukioja1992).

Preparation of midgut extracts from S. littoralis larvae

Sixth instar S. littoralis larvae were anesthetised on ice after 24 h of rearing on each of the maize hybrid diets and dissected under a stereomicroscope. Twenty larvae were randomly selected from each maize hybrid. Extraneous tissues were removed and haemolymph was rinsed with precooled, distilled water. The midguts were then added to the distilled water and homogenised on ice. The homogenates were then centrifuged at 14,000 g for 10 min at 4°C, and the clear supernatants were aspirated and stored at 20°C until completion of enzymatic assays.

Amylase activity

Amylase activity was assessed with the dinitrosalicylic acid (DNSA) method with 1% starch as a substrate in a universal buffer (10 mM succinate-glycine-2, morpholinoethan sulfunic acid) at pH 10. Midgut extract mixtures with 1% starch were incubated at 37°C for 30 min. The enzymatic reaction was stopped by adding 50 μl of DNSA reagent and heated for 15 min in boiling water. After cooling on ice, the mixture's adsorption was measured at 540 nm (Bernfeld, Reference Bernfeld1955). The amount of maltose released during the α-amylase assays was calculated using the standard curve created by using known amounts of maltose.

Protease activity

Proteolytic activity was measured using azolacasein (1.5%) as the substrate in universal buffer (50 mM sodium phosphate-borate) at pH 11. Fifty μl of midgut extract mixtures were combined with 80 μl of the substrate, which was then incubated at 37°C for 50 min. Thereafter, 100 μl of 30% trichloroacetic acid was added to the mixture, which was then centrifuged for 10 min at 14,000 g after being chilled for 30 min at 4°C. The supernatant was mixed with an equal volume of 2 M NaOH, and the absorbance was measured at 440 nm (Elpidina et al., Reference Elpidina, Vinokurov, Gromenko, Rudenshaya, Dunaevsky and Zhuzhikov2001). The Bradford (Reference Bradford1976) protein assay was also used to determine protein concentrations. Known amounts of bovine serum albumen were used to generate the standard curve.

Biochemical properties of maize hybrids

Biochemical characteristics of seeds from the various maize hybrids investigated were quantified to explore possible relationships between these variables and nutritional or enzymatic activities measured from S. littoralis. Assessment of each maize hybrid was performed in three replications and distilled water served as the negative control. All phytochemicals from the hybrids were measured using powdered seeds, which were used as the basis of each meridic diet.

The protein content of seeds was estimated using the Bradford technique. In brief, 100 μl of the homogenate was combined with 3 ml of Bradford reagent after 200 mg of the powdered seeds from each hybrid were homogenized in 10 ml of distilled water. The samples absorbance was measured at 595 nm using a standard of bovine serum albumin (Bradford, Reference Bradford1976).

The amount of starch in seeds was determined using Bernfeld's method. Powdered seeds (200 mg) from each hybrid investigated were mixed with 35 ml of distilled water before heating the samples to boiling point. Afterwards, 2.5 ml of iodine reagent (0.2% KI and 0.02% I2) was added to 100 μl of each sample, and the absorbance was measured at 580 nm (Bernfeld, Reference Bernfeld1955).

Total phenolic content was ascertained using the Slinkard and Singleton (Reference Slinkard and Singleton1997) method. The supernatants from the crude seed extracts were added to 1.5 ml of Folin-Ciocalteau reagent after being centrifuged for 15 min at 12,000 g. Thereafter, 1.4 ml of 7% sodium carbonate was added to the mixture and was allowed to sit in the dark for 30 min. The standard utilised was gallic acid. Reagents were combined with distilled water, which also served as the blank. The absorbance of standards and samples was measured at 765 nm using a spectrophotometer. For each hybrid, assays were completed in three replicates.

Total anthocyanin and flavonoid levels were quantified with the method described by Kim et al. (Reference Kim, Chun, Kim, Moon and Lee2003). Two g of maize hybrid seeds were placed in a mortar with 3 ml of acidified ethanol (1:100 acetic acid:ethanol). The seed samples were crushed and centrifuged for 15 min at 12,000 g. The extract was filtered through Whatman filter paper No. 1 and then boiled in a water bath for five minutes at 80°C. After cooling, absorbance of the extracts was measured at 415 nm for total flavonoids and 520 nm for total anthocyanins using a UV-visible spectrophotometer.

Statistical analysis

The data obtained from calculating digestive enzyme activities, nutritional indices and phytochemical content were examined for normality using the Shapiro–Wilk test before being subjected to one-way multivariate analysis of variance (MANOVA). The Tukey's HSD test was used to examine the statistical variances across means with a P < 0.01 threshold. Furthermore, a cluster analysis was carried out to find groups of maize hybrids with similar traits using the growth parameters and enzyme activity levels from S. littoralis larvae as variables. We determined the contribution of these variables to performance of S. littoralis on maize hybrids by a two-step cluster approach using Ward's minimum-variance hierarchical clustering method. The relationships between the nutritional indices or enzyme activities of S. littoralis and the biochemical properties of the maize hybrids evaluated were examined with Pearson's correlation analysis. All analyses were conducted in SPSS version 22.0 statistical software.

Results

Feeding and growth of S. littoralis

Table 1 displays the feeding performance of S. littoralis third to sixth instar larvae reared on diets from various maize hybrids. The highest consumption index (CI) index was recorded on the Oteel hybrid, while, the lowest was observed on the SC703 hybrid (F 4, 120 = 52.643; P < 0.01). The maximum values of approximate digestibility (AD) occurred on SC704, Valbum, and Oteel hybrids, while the lowest values were recorded on SC703 and Simon hybrids (F 4, 120 = 7.396; P < 0.01). The highest efficiency of conversion of ingested food (ECI) occurred on SC703, while the lowest was on Oteel (F 4,120 = 138.769; P < 0.01). The highest efficiency of conversion of digested food (ECD) was exhibited by larvae reared on hybrid SC703, while the lowest value was recorded from larvae reared on the Oteel hybrid (F 4,120 = 35.096; P < 0.01). The highest relative consumption rate (RCR) index was recorded from larvae reared on the Oteel hybrid, which was significantly higher than that observed from larvae reared on SC703, SC704 or Simon hybrids (F 4,120 = 31.211; P < 0.01). The lowest relative growth rate (RGR) was exhibited by larvae fed on the Oteel hybrid (F 4, 120 = 65.692; P < 0.01). In contrast, larvae reared on the SC703 hybrid exhibited the highest RGR (F 4,120 = 65.692; P < 0.01) (Table 1).

Table 1. Nutritional indices (mean ± SE) of third to sixth instar Spodoptera littoralis reared on various maize hybrids

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.

Means followed by different letters in the same column are significantly different (Tukey, P < 0.01).

Larval weight was greatest following rearing on the on the SC703 hybrid, and lowest on the Oteel hybrid (fig. 1) (F 4, 120 = 34.755; P < 0.01). Total mass of diet consumed was highest on SC704 and lowest on Simon (F 4, 120 = 7.820; P < 0.01). Larvae produced the greatest amount of frass on the SC703 hybrid, and significantly less frass was produced on the Valbum, Oteel, and Simon hybrids (F 4, 120 = 5.661; P < 0.01). The highest weight gain occurred in larvae that were fed on the SC703 hybrid and the lowest occurred on the Oteel hybrid (F 4, 120 = 98.424; P < 0.01). Furthermore, our results revealed that S. littoralis had the shortest larval period when reared on the SC703 and SC704 hybrids, whereas the longest larval period was obtained on the Oteel hybrid (F 4, 120 = 19.471; P < 0.01) (fig. 1).

Figure 1. (a) Mean larval weight, (b) food consumed, (c) frass produced, (d) larval weight gain, and (e) larval period of Spodoptera littoralis reared on various maize hybrids.

The greatest pre-pupal weight was observed in S. littoralis fed on the SC703 hybrid while the lowest weight occurred on the Oteel hybrid (F 4, 120 = 4.560; P < 0.01). Moreover, the highest and lowest pupal weights of S. littoralis occurred on the SC703 and Oteel hybrids, respectively (F 4, 120 = 14.589; P < 0.01) (fig. 2).

Figure 2. Pre-pupal and pupal weight (mg) of Spodoptera littoralis reared on various maize hybrids.

There were significant differences in the standardised insect-growth index (SII) and the index of plant quality (IPQ) on different maize hybrids evaluated (fig. 3). The highest SII (F 4, 120 = 14.186; P < 0.01) was quantified when the larvae were reared on the SC703 hybrid, whereas the lowest value occurred for larvae reared on the Oteel hybrid. The lowest IPQ value (F 4, 120 = 14.589; P < 0.01) was observed on the SC703 hybrid and the highest occurred on the Oteel hybrid (fig. 3).

Figure 3. Index of plant quality (IPQ) and standardised insect-growth index (SII) of Spodoptera littoralis reared on various maize hybrids.

Proteolytic and amylolytic activity in S. littoralis

Proteolytic activity was significantly higher in midguts of S. littoralis reared on SC703 than that observed in larvae reared on Oteel or Valbum hybrids (F 4, 10 = 76.89; P < 0.01). However, amylolytic activity was greatest among larvae fed on the Oteel hybrid, and was significantly higher than that observed among larvae reared on SC703 and Valbum hybrids (F 4,10 = 17.96; P < 0.01) (fig. 4).

Figure 4. General proteolytic and amylolytic activity in midgut extracts from sixth instar larvae of Spodoptera littoralis reared on various maize hybrids.

Cluster analysis

Various maize hybrids were grouped by visual inspection of the dendrogram presented in fig. 5. Individual clusters define maize hybrid phenotypes based on the responses of S. littoralis as measured by the nutritional indices, growth parameters, and enzymatic activities. Two main clusters (A and B) of S. littoralis emerged in the dendrogram with a single phenotype (hybrid SC703) in cluster B exhibiting the highest suitability to S. littoralis. Sub-cluster A1 included Valbum, Simon and SC704 hybrids, which were relatively suitable to S. littoralis. The Oteel hybrid separated into sub-cluster A2, which was a relatively unsuitable host for S. littoralis (fig. 5).

Figure 5. Dendrogram of various maize hybrids based on nutritional indices and enzymatic activities of Spodoptera littoralis reared on various maize hybrids.

Biochemical properties of different maize hybrids

The primary and secondary metabolites of maize hybrid evaluated are given in table 2. There were significant differences among the metabolites quantified; starch content was greatest in the SC703 hybrid and lowest in the Oteel hybrid (F 4,10 = 17.34; P < 0.01) (table 2). The Oteel hybrid was characterised by the lowest protein content, which was significantly lower than that recorded from all of the other hybrids investigated (F 4,10 = 29.36; P < 0.01). The SC703 hybrid exhibited the lowest phenolic content, while the Oteel hybrid was characterised by the highest total phenolic content (F 4,10 = 20.15; P < 0.01). Furthermore, total flavonoids were highest in the Simon hybrid, whereas the lowest flavonoid content was detected in the SC704 hybrid (F 4,10 = 25.18; P < 0.01). Total anthocyanin content was highest in the Oteel hybrid and lowest in the SC703 and SC704 hybrids (F 4,10 = 6.67; P < 0.01) (table 2).

Table 2. Biochemical characteristics (mean ± SE) (mg ml−1) of various maize hybrids

Means followed by different letters in the same column are significantly different (Tukey, P < 0.01).

Correlation analysis

Pearson's correlations investigating associations between consumption or growth of S. littoralis and digestive enzymatic activity on various maize hybrids are presented in table 3. Standardised insect-growth index, index of plant quality, relative growth rate, efficiency of conversion of ingested food, and proteolytic activity were positively correlated with protein and starch contents and negatively correlated with total phenolic and anthocyanin contents (P < 0.01). Furthermore, there was a significant negative correlation between the relative consumption rate of S. littoralis and protein or starch content in the various maize hybrids (table 3). Amylolytic activity was also significantly negatively correlated with both protein and starch contents of maize hybrids (table 3). In addition, total phenolic and anthocyanin contents were positively correlated with RCR and amylolytic activity (P < 0.01) (table 3). Flavonoid content measured in the various hybrids was not correlated (P > 0.05) with any of the nutritional or growth indices or digestive enzyme activities, while it was negatively correlated with the index of plant quality (table 3).

Table 3. Pearson's correlation coefficients (r) between nutritional and physiological characteristics of Spodoptera littoralis with biochemical traits of various maize hybrids

Correlations were evaluated based on Pearson's correlation test (P < 0.05).

Numbers in parenthesis represent P values.

Discussion

Insect feeding behaviour and digestive function are influenced by food quality, and these responses can serve as measures of plant tolerance to pests. The significance of plant nutritional quality, phytochemical metabolite composition, as well as plant physical properties are all well-known variables affecting plant-insect interactions (Kennedy et al., Reference Kennedy, Gould, Deponti and Stinner1987; Abedi et al., Reference Abedi, Golizadeh, Soufbaf, Hassanpour, Jafari-Nodoushan and Akhavan2020; Shishehbor and Hemmati, Reference Shishehbor and Hemmati2022). Our results indicate that while S. littoralis was able to utilise all of the maize hybrids evaluated here as hosts, there was considerable variability between hybrids in terms of their quality as resources and resultant development by S. littoralis. Previous investigations have revealed considerable variation in digestive enzyme response of S. littoralis depending on host plant quality and with regard to variation in both primary and secondary metabolites (Ladhari et al., Reference Ladhari, Laarif, Omezzine and Haouala2013; Khafagi et al., Reference Khafagi, Hegazi and Neama2016; Gacemi et al., Reference Gacemi, Taibi, Abed, M'hammedi Bouzina, Bellague and Tarmoul2019; Shishehbor and Hemmati, Reference Shishehbor and Hemmati2022; Zamani Fard et al., Reference Zamani Fard, Hemmati, Shishehbor and Stelinski2022; Hosseini Mousavi et al., Reference Hosseini Mousavi, Hemmati and Rasekh2023).

Our findings indicate that maize hybrid genotype affect S. littoralis feeding rates and development. Larval S. littoralis reared on the Oteel hybrid exhibited the highest approximate digestibility (AD value); however, it appears that a high AD index could not compensate for the low efficiency of conversion of digested food (ECD index) observed on this hybrid, which resulted in diminished larval growth. It is possible that the larval period was extended on this hybrid, increasing instar duration, where larger amounts of ingested food must be allocated to maintain metabolism (Scriber and Slansky, Reference Scriber and Slansky1981).

The efficiency of conversion of ingested food (ECI) is an indirect measure of food source quality and assesses the capacity of insects to consume food for growth and development (Batista Pereira et al., Reference Batista Pereira, Petacci, Fernandes, Correa, Vieira, Fatima da Silva and Malaspina2002). The low ECI value observed for S. littoralis reared on the Oteel hybrid indicates that this genotype was not readily converted into biomass. The total phenolic and anthocyanin contents of the maize hybrids were inversely related to quantified ECI values for S. littoralis developing on them. For example, high total phenolic and anthocyanin contents measured in the Oteel hybrid were coincident with low ECI values quantified for S. littoralis reared on this genotype.

Variation in the efficiency of conversion of digested food (ECD) can be attributed to differences in the biochemical properties of the consumed food (Nathan et al., Reference Nathan, Chung and Murugan2005). The Oteel hybrid displayed the lowest ECD value for S. littoralis larvae, indicating that this host was unfavourable for development of S. littoralis and may have increased metabolic costs associated with catabolism and excretion (Scriber and Slansky, Reference Scriber and Slansky1981). Furthermore, the duration of the feeding period in S. littoralis larvae is a significant determinant of both relative growth rate (RGR) and relative consumption rate (RCR) (Hemati et al., Reference Hemati, Naseri, Nouri-Ganbalani, Rafiee-Dastjerdi and Golizadeh2012b). Insect consumption rate is often positively correlated with insect body mass (Nathan et al., Reference Nathan, Chung and Murugan2005). The maize hybrids on which S. littoralis exhibited the lowest RCR of were SC703, SC704, and Simon, suggesting that these genotypes are nutritionally sub-optimal for this species. In contrast, the highest RGR value for S. littoralis was observed on the SC703 hybrid indicating that this hybrid caused the largest rate of weight gain. The lowest RGR value for S. littoralis larvae occurred on the Oteel hybrid and this may have been related to allocation of ingested food for maintenance of metabolism. The differences in primary and secondary plant metabolites between the various maize hybrids evaluated may explain the observed variation in the RGR index.

Our results indicate that the Oteel hybrid was a relatively poor host for S. littoralis as evidenced by the relatively long larval periods and low pupal weights observed on this genotype, which may be related to the low standardized insect-growth index measured on this genotype. In addition, the relatively low index of plant quality (IPQ) observed with the Oteel hybrid can be attributed to the relatively low pupal weight of larvae reared on this genotype.

Polyphagous herbivores can rapidly adjust profiles of their digestive enzymes in response to ingested plant PIs. This adjustment involves both the reduction and augmentation of gut proteases (Jongsma and Bolter, Reference Jongsma and Bolter1997; Hemmati et al., Reference Hemmati, Takalloo, Taghdir, Mehrabadi, Balalaei, Moharramipour and Sajedi2021). In the present investigation, our measurements focused on proteases and amylases, which constitute the principal digestive enzymes within the midgut of S. littoralis. The role of digestive proteases in insect physiology encompasses two key functions: neutralisation of protein toxins assimilated during feeding and breakdown of proteins into essential amino acids that facilitates growth and development (Nation, Reference Nation2002). The abundance of proteins in maize seeds could potentially impact major insect digestive enzymes such as proteases (Biggs and McGregor, Reference Biggs and McGregor1996). The inhibition of digestive enzymes through the presence of inhibitors results in the suppression of gut proteases and other digestive enzymes, including amylases. This suppression subsequently gives rise to suboptimal nutrient utilisation, hindered growth, and ultimately, mortality due to starvation (Hemmati et al., Reference Hemmati, Takalloo, Taghdir, Mehrabadi, Balalaei, Moharramipour and Sajedi2021).

Our results confirmed a positive correlation between proteolytic activity in the midgut of S. littoralis and the protein content present among the in maize hybrids evaluated. Proteases are likely the main digestive enzymes in the midgut of S. littoralis and these were elevated in larvae that fed on hybrids characterised by greater protein content. It is likely that feeding on protein-rich hybrid genotypes stimulates heightened production of these digestive enzymes in S. littoralis. In contrast, the amylolytic activity of larvae feeding on these hybrids exhibited a negative correlation with starch content. Notably, plants often produce amylase inhibitors that hinder the breakdown of starch molecules by digestive amylases. Remarkably, S. littoralis larvae feeding on the Oteel hybrid demonstrated the most elevated amylase activity when compared to other maize hybrids. This result may have been caused by a particularly rich store of amylase inhibitors in the Oteel hybrid, resulting in reduced starch utilisation. Consequently, this inhibition-induced scarcity of starch may have prompted an upsurge in amylase synthesis as a compensatory response to the inhibitory influence (Bouayad et al., Reference Bouayad, Rharrabe, Ghailani and Sayah2008; Hemmati et al., Reference Hemmati, Takalloo, Taghdir, Mehrabadi, Balalaei, Moharramipour and Sajedi2021).

The application of cluster analysis delineated three discrete groups of maize hybrids based on similarity of performance and enzymatic activity of S. littoralis reared on them. Cluster A1 was comprised of the Valbum, Simon, and SC704 hybrids, which can be characterised as intermediately suitable hosts of S. littoralis. The Oteel hybrid segregated from the others in clusters A or B as a relatively unsuitable genotype in sub-cluster A2. This positioning of the Oteel hybrid may be attributed to certain unique characteristics observed in larvae that developed on it including the lowest index of performance (IPQ) along with the highest values of secondary metabolites among the hybrids evaluated. Finally, cluster B was comprised of the SC703 hybrid, which is characterised as a relatively suitable genotype for development and performance of S. littoralis with reference to the evaluated parameters.

Our results confirmed an inverse relationship between the quantities of secondary metabolites among a diversity of maize hybrids and multiple nutritional and growth attributes exhibited by S. littoralis developing on them. These findings substantiate the deleterious impact of secondary compounds in these hybrids on the growth and performance of S. littoralis. Furthermore, our results suggest that the Oteel hybrid could be a useful candidate for cultivation in areas where options for chemical control of S. littoralis are limited or within the context of integrated pest management of maize fields, where plant tolerance traits are exploited as a component of the overall management program.

In summary, the present study characterised differences among several maize hybrids with respect to performance and growth of S. littoralis larvae, as well as their digestive enzyme responses after feeding. Our results indicate that the Oteel hybrid may be particularly rich in amylase inhibitors and exerted a particularly strong antibiosis effects against S. littoralis. Further research is needed to pinpoint the precise plant compound(s) responsible for the reduced growth and development of S. littoralis observed on certain hybrids such as Oteel. Identification of potential digestive enzyme inhibitors could be useful for development of resistance or tolerance in maize to chewing pests such as S. littoralis via traditional breeding or transgenic approaches. Moreover, it would be useful to investigate a broader range of plant genotypes with S. littoralis to potentially identify cultivars exhibiting even greater tolerance traits. A comprehensive exploration encompassing biochemical and molecular analyses of midgut proteases and carbohydrases could provide insights into the adaptive responses of S. littoralis larvae when exposed to various maize cultivars in their diet.

Acknowledgements

This research was funded by Shahid Chamran University of Ahvaz, Ahvaz, Iran (Grant No. SCU.AP1401.39134).

Competing interests

None.

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Figure 0

Table 1. Nutritional indices (mean ± SE) of third to sixth instar Spodoptera littoralis reared on various maize hybrids

Figure 1

Figure 1. (a) Mean larval weight, (b) food consumed, (c) frass produced, (d) larval weight gain, and (e) larval period of Spodoptera littoralis reared on various maize hybrids.

Figure 2

Figure 2. Pre-pupal and pupal weight (mg) of Spodoptera littoralis reared on various maize hybrids.

Figure 3

Figure 3. Index of plant quality (IPQ) and standardised insect-growth index (SII) of Spodoptera littoralis reared on various maize hybrids.

Figure 4

Figure 4. General proteolytic and amylolytic activity in midgut extracts from sixth instar larvae of Spodoptera littoralis reared on various maize hybrids.

Figure 5

Figure 5. Dendrogram of various maize hybrids based on nutritional indices and enzymatic activities of Spodoptera littoralis reared on various maize hybrids.

Figure 6

Table 2. Biochemical characteristics (mean ± SE) (mg ml−1) of various maize hybrids

Figure 7

Table 3. Pearson's correlation coefficients (r) between nutritional and physiological characteristics of Spodoptera littoralis with biochemical traits of various maize hybrids