Effects of Food Quality on Biology and Physiological Traits of Sitotroga cerealella (Lepidoptera: Gelechiidae)

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Plant Resistance

Effects of Food Quality on Biology and Physiological Traits of Sitotroga cerealella (Lepidoptera: Gelechiidae)

E. Borzoui, B. Naseri,

¹

and G. Nouri-Ganbalani

Department of Plant Protection, Faculty of Agriculture and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran ([email protected]; [email protected]; [email protected]), and¹Corresponding author, e-mail: [email protected] Subject Editor: Thomas Phillips

Received 9 August 2016; Editorial decision 14 November 2016

Abstract

Biology and physiological traits ofSitotroga cerealellaOlivier, a world-wide insect pest of cereals, were investi- gated on different grains (barley, maize, rye, sorghum, triticale, and wheat). Larval and pupal duration was the shortest on wheat and triticale, and the longest on sorghum. There were significant differences in survival rate of immature stages on grains with different seed hardness. The highest realized fecundity and egg fertility was observed on triticale and the lowest was seen on sorghum. Larvae fed on triticale and wheat showed higher amount ofa-amylase activity than larvae fed on other grains. MaximumVmax/KMratio was determined for the midguta-amylase ofS. cerealellalarvae fed on wheat. Whole-body protein, lipid, and glycogen contents of pupae reared on sorghum and rye were significantly lower than those reared on other grains. The statistical analysis showed that the clear correlation could be drawn between the biological characteristics and energy contents ofS. cerealellaon one side and seed hardness, amylolytic activity, and food consumed on the other.

According to the findings of this study, the variable responses ofS. cerealellato feeding on different host grains could be attributed to the quality of diets tested.

Key words:energy content, grain, life span, nutritional physiology, the Angoumois grain moth

The Angoumois grain moth,Sitotroga cerealellaOlivier, is one of the most important lepidopteran pests belonging to the family Gelechiidae. This species attacks both in field and storeroom a var- iety of seeds, with a strong preference for certain grains (Hashem et al. 2014). The larval feeding leads to considerable qualitative and quantitative losses represented as decrease of the nutritional value of the grains, failure of grain germination, and loss of seeds weight (Fouad et al. 2013b,Throne and Weaver 2013). Although some syn- thetic insecticides are the most cost-effective and widely used against S. cerealella, their use increases production costs and usually results in pest resistance as well as leads to environmental and human health risks (Peres and Corr^ea–Ferreira 2004). These reasons high- light the need for alternative control options to conventional insecticides. Plant resistance is one of the most economic means of reducing the losses due to insect pests (Parde et al. 2010). In this study, it is hypothesized that the differences in physical and nutritional characteristics of tested grains can influence the biology and digestive physiology ofS. cerealella.

Since animals obtain energy and nutrients from food, diet quality and quantity can be considered as key factors that potentially affect their life cycle traits (Karasov et al. 2011). There is a variation in plant macronutrients between and within species as a result of geno- typic differences and environmental conditions (Gall and Behmer

2014,Borzoui et al. 2015). Natural selection favors phytophagous insects that can regulate a balanced nutrient intake (Behmer 2009).

This balance depends on the interplay between food intake, digestion, and the allocation of acquired energy to various functions such as development, survival, and reproduction (Nayaa et al. 2007).

Previous studies showed that major digestive enzymes (proteases,a- amylase, and lipase) can change in proportion to the dietary content of their respective substrates (Kotkar et al. 2009,Bin et al. 2011, Borzoui et al. 2015).

Insects that feed on diets with high harmful allelochemicals gen- erally have longer developmental time and lower growth rate than those fed on diets with low harmful allelochemicals (Bin et al.

2011). Inhibition of digestive enzymes of insect pests is a part of the defensive system of host plants, as plant proteinaceous inhibitors and secondary metabolites may affect grains suitability toS. cerealella. Interference with food digestion inS. cerealellawould make it more susceptible to environmental factors (Borzoui and Bandani 2013).

Energy reserves as total protein, glycogen, and lipid content are one of the most important traits of an insect individual that greatly influence fitness characteristics, such as development and fecundity (Blanckenhorn et al. 2007). Energy reserves of insects can be considered as indicative biomarker of its overall condition, and

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variation in their concentration is used as an index of stressful conditions. Insects that experience natural cycles of food intake may have interesting adaptations for use and storage of energy sources (Sanchez–Paza et al. 2006).

The aim of this study was to examine the effect of various host grains on the biology (egg hatching, development, immature mortal- ity, and fecundity) and some physiological aspects (energy reserves and digestive activity) ofS. cerealella. Such studies may provide a better understanding of the effect of physical and nutritional characteristics of various hosts on population dynamics ofS. cerealellathat could be used in developing pest management strategies based on host plant suitability. Furthermore, study of the physiological responses ofS. cerealellato the digestivea-amylase inhibitors of the tested grains would be helpful for the selection of appropriate inhibitors and their transgenic expression for resistance to this pest.

Materials and Methods

Grains Tested

Different grains including barley (Hordeum vulgareL.), maize (Zea maysL.), rye (Secale cerealL.), sorghum (Sorghum bicolorL.), triticale (Triticale hexaploideLart.), and wheat (Triticum aestivumL.) were obtained from the Plant and Seed Improvement Research Institute (Karaj, Iran).

The experimental grain hardness was determined by standard particle size index (%) method (AACC 55-30 2000). Five replicates were carried out for each grain.

Insect Rearing

Sitotroga cerealellaadults used for the experiments were collected from stored maize seeds from Ardabil, Iran. Insect cultures were maintained on rice as a rearing host at 2561C, relative humidity of 6565%, and a photoperiod of 16:8 (L:D) h, as described by Throne and Weaver (2013)for three generations. After colonization on rice, the offsprings collected from fourth generation were used for the experiments. Moisture content of different grains was measured using the hot air oven method (AOAC 1984) and varied from 10.0% to 12.4%.

Life History Variables Development and Survival

Prior to experimentation, 500 g of each grain was held in the experimental room for 48 h. One hundred newly hatched larvae (<24 h old) ofS. cerealellawere singly transferred into one hundred Petri dishes (diameter 6 cm, depth 1 cm), containing one seed of each grain. These Petri dishes were then checked daily, and the number of adults emerging each day was recorded, allowing calculation of the duration of larval and pupal stage (from hatching egg to emergence of adult;n¼45–90), immature survival (n¼5), and adult longevity (n¼20–45 for male;n¼25–46 for female).

Realized Fecundity and Egg Fertility

One pair of newly emerged adults ofS. cerealella(one male and one female) was introduced to plastic vial (diameter 5 cm, depth 10 cm).

Black paper sheets were used as the substrate of deposited eggs (Ellington, 1930). The paper sheets were replaced daily with new ones and the number of deposited eggs was recorded every 24 h until the adult moths died (n ¼25–46). All eggs collected were monitored for 12 d to determine the number that successfully hatched (n¼25–46). These experiments were done for all tested grains.

Food Consumption

To determine food consumption (n¼5) by the whole larval stage of S. cerealella, the initial weight of each grain given to larvae and the final weight of consumed grains after adult eclosion were calculated.

Adults’ Mass

Three-hundred newly laid eggs (<24 h old) of S. cerealella were transferred into glass dishes (diameter 15 cm, depth 20 cm), containing 200 g of each tested grain. Each glass dish was monitored daily for emergence of adult moths. The male and female adults emerged (<24 h old) from insects reared on each grain were weighed separ- ately. For each sex, five replicates with 15 adults each were weighed on each host grain.

Enzyme Activity in Insects and Grains

The enzyme preparation of fourth instar ofS. cerealellafed on tested grains was based on the previously described methods (Borzoui and Naseri 2016). The midguts were homogenized at 4C for 3 min on ice-cold solution of 10 mM NaCl using a precooled homogenizer (Teflon pestle, 0.1 mm clearance) and centrifuged at 12,000g at 4C for 15 min. The supernatant was used as a source of digestive enzymes. For inhibition experiment, the enzyme extracts prepared fromS. cerealellalarvae fed on each grain were used.

The dinitrosalicylic acid (DNS) method was used to assay the digestive amylolytic activity ofS. cerealellalarvae (n¼5) fed on different grains (Bernfeld 1955) with some modifications described by Borzoui and Bandani (2013).

Kinetic studies ofa-amylase were carried out in the presence of starch concentrations (0.025, 0.5, 0.1, 0.25, 0.75, and 1%) as substrate, but the enzyme concentration was kept constant. The apparent Michaelis-Menten constant (KM) and apparent maximum velocity (Vmax) were obtained by nonlinear regression.

The proteinaceous inhibitors of the tested grains were extracted based on the methods ofBaker (1987)andMelo et al. (1999). Dry seeds were ground into flour and 30 g of the flour was extracted with 0.1 M NaCl (1:5, w/v, meal to buffer ratio) with continuous stirring at 4C for 1.5 h. Thereafter, the suspension was centrifuged at 8,000gat 4C for 30 min. The supernatant was collected and stored frozen in aliquots. Protein concentration of the inhibitors in the aliquot was determined using bovine serum albumin (BSA;

Roche Co., Germany) as standard (Bradford 1976). The effect of proteinaceous inhibitors of tested grains ona-amylase activity of fourthinstar larvae (n¼5) was studied (Baker 1987, Melo et al.

1999). The enzyme prepared fromS. cerealellalarvae fed on each grain was incubated with 1.5 mg proteinaceous inhibitor extracted from the same grain for 10 min prior to the addition of substrate.

The procedure for the amylolytic assay was conducted as described earlier. For the control group, the same enzyme extract was used without inhibitor. The enzyme inhibitory activity is expressed as the decrease in percentage of maltose liberated. The percentage of a-amylase inhibition was calculated according to Borzoui and Bandani (2013).

Biochemical Analyses of Pupae

For the biochemical analysis,S. cerealellapupae that came from larvae fed on each grain were singly cold-anesthetized at 4C.

Subsequently, whole-body protein (n ¼5), glycogen (n¼5), and lipid (n¼5) contents were determined. Five replicates were per- formed for each grain. The protein content was analyzed using Lowry reagent as described byLowry et al. (1951)and quantified by BSA as standard. The lipid content was measured by the method of

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Van Handel (1985) using sulphosphovanillin reagent (orthophos- phoric acid 0.6% aqueous vanillin solution 4:1 v/v; Gessner and Neumann 2005) after extraction of lipid by using chloroform:

methanol solution (1:1 v/v). The lipid content was quantified by comparison to a cholesterol palmitate standard curve. The glycogen content was estimated using anthrone reagent as described by Bemani et al. (2012)and quantified by standard curve that was prepared using glycogen. The amounts of protein, glycogen, and lipid were expressed asmg/pupa.

Statistical Analyses

All the data were analyzed using SAS ver.9.2 program (PROC GLM;SAS Institute 2009). One-way analysis of variance (ANOVA) was used to examine the effect of the grains on life history, amylolytic activity, and energy contents ofS. cerealella. The statistical differences among the means were compared using LSD (least significant difference) test ata¼0.05. The Pearson correlation coef- ficient was used to investigate the relationships between some physiological and life history traits and body energy contents of S. cerealella fed on different grains and amylolytic activity, seed hardness, and food consumed by larvae using SPSS 16.0.

Results

Effects of Grain on Life History

The developmental time (larval and pupal period) and adult longevity ofS. cerealellavaried significantly on different grains (Table 1).

The longest developmental time (F_{5, 416}¼157.58,P<0.001) was on sorghum and the shortest was on wheat and triticale. Moreover, male (F5, 198 ¼31.62, P<0.001) and female (F5, 215 ¼35.40, P<0.001) longevity was significantly affected by different grains fed by larvae. The records for male and female longevity were longest whenS. cerealellawas reared on wheat and triticale, and were shortest when it was fed on sorghum.

Different grains significantly affected fecundity (F_{5, 212}¼240.09, P<0.001) and fertility (F5, 212¼221.43,P<0.001) ofS. cerealella (Table 1). The greatest number of eggs laid was from females reared on triticale, and the least number was from females reared on sorghum and rye. Also, among six grains tested, the highest egg hatch- ability (fertility) was recorded on triticale and wheat, and the lowest was obtained on sorghum and rye.

Significant differences were observed in the survival rate of S. cerealellaimmature stages after feeding on different grains (Fig.

1). The survival rate of immature stages (F5, 24¼36.09,P<0.001) was the highest on triticale and wheat, and the lowest on sorghum.

Figure 2shows the food consumed byS. cerealellalarvae on different grains (F5, 24¼60.54,P<0.001). The larvae reared on maize consumed more food than those reared on other grains. In contrast, larvae reared on rye ate less food than those reared on the others.

Significant differences were seen in adults’ mass ofS. cerealellafed on different grains (F5, 30¼21.75,P<0.001 for male;F5, 30¼70.97, P<0.001 for female; Fig. 3). Male adults who came from larvae reared on triticale and wheat were heavier than those reared on other grains. Moreover, female adults reared on triticale, wheat, and maize were heavier than those reared on other grains. By contrast, male and female adults who came from larvae reared on sorghum were lighter than those reared on other tested grains.

Midgut Enzyme Activity

Tested grains significantly affected digestivea-amylase activity in larvae ofS. cerealella(F5, 24¼115.41,P<0.001). The amylolytic activity in midgut of fourth instar was higher on triticale and wheat than on other grains. However, amylolytic activity was the lowest when larvae were reared on sorghum (Fig. 4).

The results of this study showed that thea-amylase of fourth instar reared on different grains had various kinetic parameters against starch (Table 2). TheKMvalue for thea-amylase of larvae was the highest on sorghum and the lowest on triticale and wheat.

Also, among tested grains, wheat showed the maximum reaction rate (Vmax) value as 0.53 mmol/min/mg protein. Moreover, max- imumVmax/KMratio was determined for midguta-amylase of S.

cerealellalarvae fed on wheat.

Table 1.Mean (6SE) duration (d) of larval and pupal period, adults longevity, realized fecundity (eggs laid per female), and egg fertility (percentage of hatched eggs per female) ofS. cerealellafed on different grains

Diet n Larval and pupal period n Male longevity n Female longevity n Fecundity n Fertility (%) Barley 67 25.1060.18bc 34 7.6460.16b 33 8.0060.16bc 33 79.161.3c 33 74.166.5c

Maize 74 24.9760.25c 35 7.5160.21b 39 8.3060.23b 39 76.461.3c 39 77.262.2b

Rye 57 25.8560.27ab 26 6.5060.19c 31 7.3260.18cd 31 48.261.1d 31 62.467.4d

Sorghum 45 26.2660.24a 20 5.4560.21d 25 6.4860.23d 25 45.161.0d 25 59.065.4d Triticale 90 21.8160.11d 44 8.6560.18a 46 9.6560.19a 46 100.661.2a 46 93.664.2a

Wheat 89 21.7460.15d 45 8.4260.17a 44 9.4460.16a 44 93.161.8b 44 90.964.5a

The means followed by different letters in the same column are significantly different (LSD,P<0.05).

Thenvalue shows the number of insects tested for each parameter.

Fig. 1.Mean (6SE) percentage survival ofS. cerealellaimmature stages on different grains. Each point is average of five replications. The means followed by different letters are significantly different (LSD,P<0.05).

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Figure 5 shows the effect of the proteinaceous inhibitors extracted from different grains on thea-amylase activity of fourth instar of S. cerealella (F5, 24¼16.11, P<0.001). The highest inhibition was recorded for the proteinaceous inhibitor extracted from sorghum. However, the inhibitor extracted from maize showed the lowest inhibitory effect againsta-amylase of this pest.

Pupal Nutrient Analyses

There was a significant difference for the energy contents of S. cerealellapupae came from larvae reared on different grains (F5, 24¼41.49,P<0.001, for protein;F5, 24 ¼38.94,P<0.001, for lipid;F_{5, 24}¼7.81,P<0.001, for glycogen;Table 3). Whole-body protein, lipid, and glycogen contents of pupae on sorghum and rye were significantly lower than those on the other grains. The mean protein and glycogen levels of triticale- and wheat-fed insects were significantly higher than the other grains. The pupae that came from larvae fed on maize, triticale, and wheat showed the highest lipid content; however, the lowest content was recorded for pupae that came from larvae reared on rye and sorghum.

Grain Hardness

Particle size index (PSI; as an indicator of grain hardness) of host grains ranged from 11.7% in maize to 33.1% in triticale (Fig. 6).

Lower PSI values for maize and sorghum grains show that they are harder than the others. By contrast, higher PSI values for triticale and wheat indicate that these grains are softer than the others.

Correlation Analyses

Table 4 shows the correlation coefficients analysis of the physiological and life history traits and body energy content ofS. cerealella fed on different grains with amylolytic activity, seed hardness, and food consumed. The results of this study showed that very high correlations existed between the biological characteristics and energy contents on one side and amylolytic activity on the other. Also, significant positive correlations were found between weight of male and female (r¼0.736 and 0.791, respectively) and protein, lipid, and glycogen contents (r¼0.733, 0.737, and 0.761, respectively) with food consumed. Moreover, immature period, survival, and amylolytic activity exhibited a significant correlation with seed hardness (r¼ 0.715, 0.781, and 0.817, respectively).

Discussion

Eating a low-quality food has been associated with reduced growth and weight, delayed development, and low survival rate output in insect herbivores (Lee 2007). In this respect, we explored if the features of food modify life history, body energy content, and digestive physiology ofS. cerealella. Our results showed that the development of larval and pupal stages on rye and sorghum was significantly prolonged compared to those reared on wheat, triticale, maize, or barley. There are two possible explanations for this observation. First, the difference of immature period seems to be mainly due to the differences of essential nutritional factors or physical properties of the tested grains.

Second, the insects reared on rye and sorghum may utilize ingested nutrients less efficiently than that other fed on other grains.

Correlation coefficients given inTable 4between immature stages with amylolytic activity and PSI support this finding. In a similar study,Hamed and Nadeem (2012)found such difference in the developmental time ofS. cerealellaon different grains.

In this study,S. cerealellafed on wheat and triticale showed the longest adult longevity, suggesting that these grains had relatively optimal conditions for better development of larvae, which led to the emergence of adults with longer longevity. The adult longevity ofS. cerealella, in this study, agreed with the results of other reports (Hansen et al. 2004,Saikia et al. 2014).

Survival rate of immature stages was the greatest inS. cerealella fed on triticale and wheat, and it is similar to data reported from survival rate ofS. cerealellareared on different hosts under similar conditions (Wongo 1998, Shafique et al. 2006). Greenberg et al.

(2001) suggested that shorter developmental time and higher sur- vivorship ofS. exiguacan lead to higher values of growth index of this pest. According to this viewpoint, triticale and wheat are more suitable with better quality compared to the other grains tested.

The present study shows that quality of larval diet had significant effect on the fecundity and fertility ofS. cerealella. In most grains, there was a direct correlation between fecundity (r¼0.913) and fertility (r¼0.938) and larval amylolytic activity. Moreover, the amylolytic activity exhibited a high correlation with PSI (r¼817). It is suggested that less hardening of grains led to higher amylolytic activity and more comfortable consumption of food by larvae.Fouad et al.

(2013a) found that seed hardness is important factor for the determination of susceptibility of corn seeds to infestation byS. cereallela.

High fecundity and fertility resulting from better quality of food would increase the number of offspring contributing to population growth from generation to generation.

In this study, among the tested grains, the midgut amylolytic activity in fourth instar ofS. cereallelafed on triticale and wheat was higher than the amylolytic activity of larvae fed on barley, maize, rye, and sorghum. Different levels of amylolytic activity can be posi- tively correlated to possible structural differences of the examined grains. Also, high amylase activity in the triticale- and wheat-fed larvae might be due to the nutritionally balanced compositions of these diets. A major difference was reported in midgut amylolytic activity ofSitophilus granariusL. (Col.: Curculionidae) fed on cereal grains (Piasecka–Kwiatkowska et al. 2014). Despite various activities of digestivea-amylase of larvae on different grains, a higher correlation was found between amylolytic activity and the developmental time, survival rate, fecundity, and fertility ofS. cereallela. Larvae feeding on the grains that were more suitable forS. cerealleladevelopment (triticale and wheat) had higher amylolytic activity, which was also supported byKotkar et al. (2009)andBorzoui et al. (2015). This trade-off between enzyme activity and nutritional composition of Fig. 2.Mean (6SE) food consumed byS. cerealellalarvae on different grains.

Each point is average of five replications. The means followed by different letters are significantly different (LSD,P<0.05).

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food is consistent with the endocrinological control model of insect development (Behmer 2009,Karasov et al. 2011).

TheKMvalue determines the affinity of enzyme for the substrate (Kaur et al. 2015). In this study, the specific action ofS. cereallelaa-

amylase reared on different grains was reflected in theKMvalues determined for starch. TheKMvalues fora-amylase of larvae fed on triticale and wheat were lower than those fed on the other grains.

LowerKMvalues may be attributed to an increased affinity of the substrate for the enzyme. In addition,Vmaxreveals the number of starch molecules converted into product bya-amylase in a unit time when this enzyme is fully saturated with starch (Kaur et al. 2014). In the present study, whenVmaxvalues ofa-amylase of larvae reared on different grains were compared, the highest and lowest levels of hydrolysis were observed for larvae fed on wheat and sorghum, respectively.Ozgur et al. (2009)reported that the decrease inVmax

demonstrates a decrease in the rate of hydrolysis of starch. Based on KMandVmaxvalues, it can be suggested thatS. cereallelalarvae pro- duced different isoenzymes ofa-amylase with different properties in response to feeding on different grains. On the other hand, catalytic efficiency (Vmax/KM ratio) ofa-amylase of this pest is higher on wheat than on other tested grains. We suggest that thea-amylase of S. cereallela larvae tends to operate on starch reserves of wheat more efficiently than on starch reserves of other tested grains.

Fig. 3.Mean (6SE) weight of male and female adults ofS. cerealellafed on different grains. Each point is average of five replications. The means followed by different letters are significantly different (LSD,P<0.05).

Fig. 4.Mean (6SE)a-amylase activity of midgut extracts fromS. cerealella larvae fed on different grains. Each point is average of five replications. The means followed by different letters are significantly different (LSD,P<0.05).

Table 2.Apparent Michaelis–Menten constant (K_M), apparent maximum velocity (V_max), andV_max/K_Mratio fora-amylases from the midgut of S.cerealellalarvae fed on different grains against the starch substrate

Diet KM(%) Vmax(mmol/min/mg protein) Vmax/KMratio

Barley 0.038 0.42 11.0

Maize 0.024 0.39 16.2

Rye 0.040 0.32 8.0

Sorghum 0.056 0.24 4.3

Triticale 0.023 0.51 22.2

Wheat 0.023 0.53 23.0

Fig. 5.Mean (6SE) percentage inhibition of amylolytic activity ofS. cerealella larval midgut by crude inhibitor extracted from different grains. Enzyme and inhibitor were incubated for 30 min prior to the addition of starch to measure enzymatic activity. Each point is average of five replications. The means followed by different letters are significantly different (LSD,P<0.05).

Table 3.Energy reserves (mg/pupa) ofS.cerealellapupae that came from larvae fed on different grains

Diet Energy content (mean6SE)

Protein Lipid Glycogen

Barley 184.8613.2b 361.6611.6b 54.265.2bc Maize 208.8614.7ab 387.0614.7a 59.065.9ab Rye 150.2612.8c 333.4611.1c 49.262.0c Sorghum 141.2615.0c 323.4610.0c 47.465.9c Triticale 229.2610.7a 406.6611.4a 61.864.2a Wheat 223.6610.8a 405.2616.9a 61.864.3a The means followed by different letters in the same column are significantly different (LSD,P<0.05),n¼5.

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Plant proteinaceous inhibitors are natural control agents against herbivorous insects, because they reduce digestive enzymes activity and affect developmental characteristics of different species of Coleoptera and Lepidoptera (Farias et al. 2007,Chougule et al. 2008).

The results of this study demonstrate that the impairment of the digestive process ofa-amylase occurs after incubation with the inhibitors. Our results corroborate previous studies in which inhibitory activity was reported for barley (Zhang et al. 1997), maize (Kumari et al. 2012), rye (Iulek et al. 2000), sorghum (Kotkar et al. 2009), triticale (Mohammadzadeha et al. 2013), and wheat (Borzoui and Naseri 2016). However, these earlier studies found the different effect of these inhibitors on the amylolytic activity. This led to the conclusion thatS.

cereallela larvae adapted to some proteinceous inhibitors of tested grains, as noted byLomate and Hivrale (2011).

Typically, when the amount of food consumed decreases, the insects developmental time is extended and their body weight decreases (Phillipson 1981). In our study, the amount of food consumed was significantly different when larvae were fed on different diets,

suggesting that the quality of food consumed might have a main effect on fitness ofS. cereallela. The high levels of maize consumed byS.

cerealellain our experiments (Fig. 2) may be due to the hardness of maize (Morris 2002). On the other hand, although maize grain was harder than the other tested grains; however, the inhibitor extracted from it showed the lowest negative effects againsta-amylase of larvae (Fig. 5), leading to increased food consumed by larvae, and body weight of female adults reared on this host grain.

The effect of different grains onS. cereallelawas most obvious in pupal energy contents (Table 3). These biomarkers are reflection of the entire physiological status of the insect and a measure of its energy budget, which can be affected by food consumed in larval stage (Laparie et al. 2012). We observed the lowest protein, glycogen, and lipid contents in pupae from larvae that had fed on sorghum, which had the lowest amylolytic activity. It is reasonable to conclude that a decreased body energy contents can be achieved by the reduced amylolytic activity (Fig. 4) and greater hardness of sorghum grain (Fig. 6). High correlations between immature period and amylolytic activity (r¼ 0.928) on one side, and amylolytic activity and PSI (r¼0.817) on the other support this hypothesis. However, if the seed hardness is a unique factor that affected energy contents of S. cereallela, then insect fed on maize would have the lowest energy contents. The observed data did not confirm this hypothesis.

The possible explanation for this could be that when the level of a-amylase inhibitors is low, it seems that the insect is partly able to overcome increased seed hardness.

Intraspecific body weight variation is often related to the amount of reserves that individuals can store, or have already stored (Rion and Kawecki 2007). In this study, different diets had significant effect on the adult body weight ofS. cerealellaand in this respect our results are comparable to those found forS. cerealellabyHamed and Nadeem (2012). Larvae reared on sorghum showed reduced body weight in the adult stage. The results suggest that the food quality is a key factor during the larval phase, as duration of development increased in larvae reared on a grain of lower quality.

Blanckenhorn (2000) reported that some counterbalancing forces may keep organisms small, as achieving large body size includes po- tential costs, such as high energetic requirements for maintenance and prolonged larval stages.

In conclusion, the results of this study clearly demonstrate that differences in food quality among different grains can play a key role inS. cereallelafitness. The longest developmental time, the lowest adult weight, fecundity, and fertility were forS. cerealellafed on sorghum, which is a hard grain with the highest level ofa-amylase inhibition. According to our data, we conclude that seed hardness together with the level of proteinaceous inhibitors resulted in considerable negative effects on the amylolytic activity, which led to the significant reduction in fitness ofS. cereallela. Correlation coefficients analysis showed that amylolytic activity correlates to seed hardness; it seems that there exists an insect mechanism to precisely detect the seed physical characteristics and adjust the levels of these important digestive enzymes. Also, pupae from larvae fed on the different grains were significantly different in energy contents. To understand the resistance factors of tested grains toS. cerealella, it is important to characterize not only proteinaceous inhibitors and seed hardness of the hosts but also the susceptibility ofS. cerealellato other physical and biochemical characteristics of these grains and other hosts. According to the results of this study, sorghum and rye were identified as unsuitable hosts, and triticale and wheat were identified as suitable hosts for feeding and development of S. cerealella.

Fig. 6.Comparison of seed hardness of the different tested grains by using particle size index (%).

Table 4.Correlation coefficients (r) of some biological and physiological characteristics ofS.cerealellafed on different grains with amylolytic activity, seed hardness, and food consumed

Parameter Amylolytic

activity

Particle size index

Food consumed

r Pvalue r Pvalue r Pvalue

Immature period 0.928 0.002 0.715 0.034 0.437 0.153 Male longevity 0.907 0.003 0.575 0.081 0.490 0.122 Female longevity 0.932 0.002 0.633 0.059 0.522 0.105 Fecundity 0.913 0.003 0.578 0.079 0.527 0.102 Fertility 0.938 0.001 0.635 0.058 0.542 0.095 Survival 0.985 0.000 0.781 0.020 0.378 0.194 Weight of male 0.753 0.020 0.360 0.208 0.736 0.029 Weight of female 0.705 0.037 0.305 0.256 0.791 0.018 Protein content 0.811 0.014 0.416 0.166 0.733 0.030 Lipid content 0.818 0.013 0.430 0.157 0.737 0.029 Glycogen content 0.797 0.017 0.400 0.178 0.761 0.023 Amylolytic activity – – 0.817 0.013 0.313 0.248

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Acknowledgments

We thank the University of Mohaghegh Ardabili, (Ardabil, Iran), for cooper- ation by support for the experiment.

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