Layout 1 Abstract The effects of four mulberry varieties (Kenmochi, Ichinose, Shin Ichinose, Mahalii) on nutritional indices and digestive proteolytic and amylolytic activities of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) were determined at 24±1°C, 75±5% RH and a photoperiod of 16:8 L:D. Fifth instar larvae reared on Shin Ichinose showed the highest efficiency of conversion of digested food and efficiency of con- version of ingested food (3.82±0.16% and 3.11±0.07%, respectively). Approximate digestibility values of the fourth instar larvae were high- est (95.23±0.73%) and lowest (91.77±1.45%) on Kenmochi and Shin Ichinose, respectively. The fifth instar larvae fed on Kenmochi had the highest consumption index (4.6±0.73) and lowest relative growth rate (0.03±0.10), respectively. Our results showed that the highest pro- tease activity in optimal pH was on Malalii variety (0.97 U/mg) and the lowest was on Kenmochi (0.75 U/mg). In addition, the highest amylase activity in optimal pH was on Mahalii (0.17 U/mg) and lowest on Kenmochi (0.103 U/mg). Specific proteolytic analysis showed that lar- vae feeding on Mahalii had the highest activity of trypsin and elastase (2.30 and 2.13 U/mg, respectively). This research showed that plastic- ity in food utilization and enzyme activity is functionally relevant to host plant cultivars. The results of nutritional indices and activity of digestive enzymes indicated that Kenmochi was an unsuitable host for feeding of Glyphodes pyloalis. Introduction Mulberry is the sole host for silkworm (Bombyx mori L) rearing and is also used for shade trees in cities (Kumar et al., 2002). The less- er mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is considered a serious pest of mulberry in India, China, Korea, Japan, Malaysia, Pakistan, Uzbekistan and Burma (Madyarov et al., 2006). This pest has caused severe damage to mulberry plantings in northern Iran and has turned into a serious concern for the silk industry (Khosravi & Sendi, 2010). Larvae form threads on the outer part of mulberry leaves and feed on the mesophyll from under those threads, leaving only a network of epidermis (Aruga, 1994). If leaves infected with excreta of larvae are fed to silkworms, they develop con- stipation and are unable to defecate (Aruga, 1994). In addition, G. pyloalis larvae are considered alternate hosts of Bombyx densoviruses and picornaviruses (Watanabe et al., 1988) Nutrition is the interaction of physiological processes and ecology, so it is directly associated with natural selection as well as competition for food (Sheikher et al., 2001; Zhu et al., 2005; Xue et al., 2010). In numerous studies of the relationships between insect pests and plants, attempts have been made to quantify the efficiency with which insects use their food plants (David & Gardiner, 1962; Sheikher et al., 2001; Xue et al., 2010). Together with data on the rate of food ingestion and growth, food utilisation efficiency is an important component of herbivore performance (Slansky & Scriber, 1985). The quality and quantity of food consumed could affect the growth, development, and reproduction of insects (Scriber & Slansky, 1981). Of the tools of pest management, host plant resistance is important in terms of being both economically and environmentally acceptable. Therefore, as a method of controlling pest insects, host plant resistance is not only favourable to the environment, but also reduces expenses for growers (Li et al., 2004). The factors determining nutrient availability for growth and maintenance over a given period of development are the amount and type of food consumed and the efficiency with which it is utilized (Barton Browme & Raubenheimer, 2003). The study of host plant resistance can play an important role in identifying anti-digestive or antifeedant compounds and their further use in pest management strategies (Lewis et al.,1997). A possible strategy to control insect pests is to produce crops with elevated levels of endogenous resistance. One such approach has been to over- express plant proteins that are known to play a role in plant defence against herbivores (Hosseininaveh et al., 2007). Many plants respond to insect feeding by the synthesis of protease inhibitors (PIs) (Ryan, 1990). Plant PIs have been shown to inhibit gut proteases of insects and prevent larval growth and development when delivered in artificial diets (Johnston et al., 1993) or when expressed in transgenic crops (Hilder, 1987). Efforts are being made to explore their use in develop- ing insect resistance in susceptible crop plants (Sharma et al., 2000). However, as a first step to achieving this goal, an understanding of how Correspondence: Jalal Jalali Sendi, Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, 416351314, Iran. Tel.: +981316690817 - Fax: +981316690281. E-mail: jjalali@guilan.ac.ir Key words: Glyphodes pyloalis, feeding indices, digestive enzymes. Received for publication: 2 May 2013. Revision received: 9 November 2013. Accepted for publication: 25 November 2013. ©Copyright M. Oftadeh et al., 2014 Licensee PAGEPress, Italy Journal of Entomological and Acarological Research 2014; 46:1633 doi:10.4081/jear.2014.1633 This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 3.0) which permits any noncom- mercial use, distribution, and reproduction in any medium, provided the orig- inal author(s) and source are credited. Effect of four varieties of mulberry on biochemistry and nutritional physiology of mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) M. Oftadeh, J.J. Sendi, A. Zibaee, B. Valizadeh Department of Plant Protection, University of Guilan, Rasht, Iran [page 42] [Journal of Entomological and Acarological Research 2014; 46:1633] Journal of Entomological and Acarological Research 2014; volume 46:1633 No n- co mm er cia l u se on ly digestive enzymes function is essential to plan strategies for success- ful and sustainable implementation of PI-based control methods (Franco et al., 2002). Insect digestive proteases catalyze the release of peptides and amino acids from dietary proteins in the insect digestive canal to meet its nutritional requirements (Terra & Ferreira, 1994). The lepidopteran larval midgut has been shown to have complex proteolytic activities including trypsins, chymotrypsins, elastases, cathepsin-B-like proteases, aminopeptidases, and carboxypeptidases that are all required in protein digestion. Lepidopteran insects mainly depend on serine proteases for protein digestion (Bown et al., 1997). The other important classes of digestive enzymes from these insects include a-amylases, which are also the main digestive enzymes of many other insects that feed absolutely on starchy seeds throughout their life (Pereira et al., 1999). The a-amylases (a-1, four glucan four glucanohydrolases; EC 3.2.1.1) catalyze hydrolysis of a -D-(1, 4)-glucan linkage in starch components glycogen, and other different associated carbohydrates to supply an energy source (Franco et al., 2000). In insects, the activity of digestive enzymes such as proteases and a-amylases depends on the nature of food sources or chemical com- pounds ingested (Mendiola-Olaya et al., 2000). Protease and a-amylase activities in crude extracts of larval guts of different lepidopteran species have been described (Zibaee et al., 2008). Study of the insect digestive system is an important method for dis- covering new control techniques in integrated pest management pro- grams (Lawerence & Koundal, 2002). Many factors are involved in the host preference of insects. Among the most important are plant species and regional plant diversity (Davidson et al., 2001), chemical composi- tion of leaves (Foss & Riseke, 2003), and leaf age, which itself is the cause of physical and chemical changes (Meyer & Montgomery, 2004). The goal of this research was to compare nutritional indices and activity of digestive enzymes in G. pyloalis larvae reared on different host plants, and to determine how these parameters change after an additional instar and in response to different hosts. Materials and methods Plant sources Four host plants were used in this study, including Kenmochi, Ichinose, Shin Ichinose and Mahalii. These varieties were selected because they are the most important economic varieties used in Iran to rear silkworms. Insect rearing G. pyloalis larvae were collected from mulberry orchards near the city of Rasht in northern Iran. They were reared on fresh mulberry leaves (different host plants) in the laboratory at 24±1°C, 75±5% RH and 16:8 (L:D) h photoperiod in transparent plastic boxes (18×15×7 cm) covered with muslin for aeration. As adults emerged, they were separated and placed in transparent plastic boxes (18×7 cm) with a 10% honey solution on cotton wool for feeding, and mulberry leaves were provided for oviposition. Nutritional indices Nutritional indices were determined using second to fifth instars, which were easier for measuring these indices than on the 1st instar. Ten newly emerged second instar larvae that had been reared on each of the four varieties of mulberry as host plants were selected and pro- vided with 10 fresh leaves from each of the four hosts; they were indi- vidually weighed and maintained in small plastic tubes (2×5 cm) until they stopped feeding before molting to the next instar. This method was used for other larval instars as well. The initial fresh food and the food and feces remaining at the end of each experiment were weighed daily. The quantity of food ingested was determined by subtracting the diet remaining at the end of each experiment from the total weight of diet provided. The weight of feces produced by the larvae fed on each mulberry variety was recorded daily. To find the dry weights of the pods, feces, and larval to adult stages, extra specimens (20 specimens for each) were weighed, oven-dried (48 h at 60°C), and then re-weighed to establish a percentage of their dry weight. Food utilization rates were then calculated based on the following formulas of Waldbauer (1968): i) consumption index (CI): calculated on the basis of the rate of intake relative to the mean weight of the larva during the feeding period, according to the following formula: CI=F/TA; ii) relative growth rate (RGR): calculated as: RGR=G/TA; iii) efficiency of conversion of ingested food (ECI): the efficiency of con- version of ingested food to body substance is calculated as: ECI=WG/FI×100; iv) approximate digestibility (AD): the approximate digestibility is calculated as: AD=FI-WF/FI×100; v) efficiency of con- version of digested food (ECD): the efficiency with which digested food is converted to body substance is calculated as: ECD=WG/FI- WF×100. Where is A=mean fresh weight of the larva during the feeding peri- od, F=fresh weight of food eaten, T=duration of feeding period (days), G=fresh weight gain of the larva during the feeding period, WG=weight gained, FI=weight of food ingested, WF=weight of feces. Biochemical assessments The guts of fifth instar larvae were used to measure proteolytic and amylolytic activities, and the whole body to measure other biochemical assessments as follows: fifth-instar larvae reared on different hosts were cold anesthetized and quickly dissected under a stereomicro- scope. The midguts were then cleaned by deletion of unneeded tissues. The midguts, including contents, were collected into a known amount of distilled water and homogenized with a handheld glass grinder on ice, and the homogenates were centrifuged at 16,000× g for 10 min at 4°C. The resulting supernatant was stored at –20°C for later protease and amylase assays. Each biochemical analysis was repeated 3 times. General proteolytic activity present in the midgut of G. pyloalis lar- vae fed on different hosts was determined using azocasein as a sub- strate at an optimum pH. A universal buffer system (50 mM sodium phosphate-borate) was used to determine the pH optimum of proteolyt- ic activity over a pH range of 7-12. To determine the azocaseinolytic activity, the reaction mixture containing 80 mL of 1.5% azocasein solu- tion in 50 mM universal buffer (pH 12) and 50 mL of crude enzyme was incubated at 37°C for 50 min. Proteolysis was terminated by the addi- tion of 100 mL of 30% trichloroacetic acid (TCA) followed by cooling at 4 °C for 30 min and centrifugation at 16,000 × g for 10 min. An equal volume of 2 M NaOH was added to the supernatant, and the absorbance was measured at 440 nm. Appropriate blanks in which TCA had been added prior to the substrate were prepared for each assay. Unit activity was expressed as an increase in optical density mg−1 protein of the tis- sue min−1 due to azocasein proteolysis (Vinokurou et al., 2007). Digestive trypsin-, chymotrypsin- and elastase-like activities of the larvae fed on either all varieties of mulberry were estimated using final concentrations of 1 mM BApNA, 1mM SAAPFpNA and 1 mM SAAApNA as substrates, respectively. A reaction mixture consisted of 20 mL of enzyme extract for trypsin- and elastase-like activities and 10 mL of enzyme extract for chymotrypsin-like activity, 75 mL of universal buffer at the appropriate pH optimum (pH 10.5 for trypsin- and chymotrypsin- like enzymes and pH 11 for elastase-like enzyme) and 5 mL of the above-mentioned substrate. Absorbance was then measured at 405 nm for 40 min (at 2, 1 and 4 min time intervals respectively). All assays were carried out in triplicate against appropriate blanks. The a-amylase activity was measured using the procedure of Bernfeld (1955), with 1% soluble starch as substrate. A quantity of 50 [Journal of Entomological and Acarological Research 2014; 46:1633] [page 43] Article No n- co mm er cia l u se on ly [page 44] [Journal of Entomological and Acarological Research 2014; 46:1633] mL of the enzyme was incubated with 250 mL of universal buffer (pH 10) and 20 mL of soluble starch for 30 min at 37°C. The reaction was terminated by addition of 50 mL DNS and heating in boiling water for 10 min. The absorbance was then read at 540 nm after cooling on ice. One unit of amylase activity was defined as the amount of enzyme required to produce 1 mg of maltose in 30 min at 37°C under the given assay conditions. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using Thomas’ (1998) procedure. This assay was done by AST and ALT kit (Biochem Co., Tehran, Iran). The absorption was read at 340 nm. Assays of estimation of acid (ACP) and alkaline phosphatases (ALP) were carried out as described by Bessey et al. (1946). The buffered sub- strate (phosphate buffer, 0.02 m, pH 7.2) was incubated with the samples for 30 min. Alkali was added to stop the reaction and to adjust the pH for the determination of concentration of the product formed. The spectral absorbance of p-nitrophenolate was maximal at 310 nm. The molar absorbance of p-nitrophenolate at 400 nm is about double that of p-nitro- phenyl phosphate at 310 nm. On converting the p-nitrophenolate into p- nitrophenol by acidification, the absorption maximum is shifted to about 320 nm with no detectable absorption at 400 nm. Glucose was analyzed as described by Sigert (1987). Protein was measured based on Bradford’s (1976) method and by utilizing a total protein assay kit (Biochem Co.). In this method, proteins made a com- plex purplish blue with an alkaline copper solution, for which the absorption value was read at 540 nm. To measure the total cholesterol of hemolymph, Richmond’s (1973) method was conducted. The principles of this method are based on hydrolysis of cholesterol esters by cholesterol oxidase, cholesterol esterase and peroxidase. Glycogen and trehalose content were assessed using the method of Van Handel (1965). This method separates glycogen and trehalose. Trehalose and glycogen were similarly assayed using the anthrone-sul- furic acid method. Chemical analysis of the leaves Leaves from all four mulberry trees were collected during the experimental period in July 2012, dried in the shade, and prepared for chemical analysis. For phosphorous, calcium and potassium, dry ashing and mixing with HCl was first performed. Total organic nitrogen con- tent was determined by the micro-Kjeldahl method, potassium content was measured by flame photometry, using a lithium internal standard, and phosphorus content was determined using the colorimetric method with blue coloured acid ascorbic and read at 880 nm. For measuring cal- cium, the compleximetric method was used. Statistical analysis Nutritional indices of G. pyloalis reared on different hosts were analyzed with one-way ANOVA using the statistical software Minitab ver. 14.0 (Minitab Inc., Philadelphia, PA, USA. http://www.minitab.com; 1994) to determine similarities and significant differences. Statistical differences among the means were assessed using an LSD test at a=0.01. Data were tested for normality before analysis. The data from other experiments were subjected to analysis of variance (ANOVA) using SAS software. The least significant differences among treat- ments were compared using Tukey’s multiple range test (SAS Institute, Cary, NC, USA; 1997). Differences among means were considered sig- nificant at P≤0.01. Results Nutritional indices The results of the nutritional indices of second- fifth larval instars of G. pyloalis are shown in Tables 1-4. Nutritional indices of the second instar larvae of G. pyloalis were significantly different for different host plants. The larvae reared on Kenmochi showed the highest value of ECD (F=17.70; df=3; P<0.0007) (1.15±0.12%) and CI (F=143.38; df=3; P<0.0001) (7.14±0.9). However, the lowest value of ECD (F=0.91; d=3; P<0.0004) and CI (F=0.35; d=3; P<0.0007) was on Ichinose (0.45±0.20% and 3.28±0.04, respectively). Also, the highest value of ECI (F=18.91; d=3; P<0.0005) (1.13±0.12%) was on Kenmochi com- pared with the other hosts. However, the larvae reared on Ichinose had the highest value of AD and the lowest value of CI (98.00±0.91 and 3.28±0.04, respectively). The highest and lowest value of RGR (F=18.6; Article Table 1. Nutritional indices of second instar larvae of Glyphodes pyloalis on different hosts (means±SE). Host CI AD ECI ECD RGR Ichinose 3.28±0.04d 98.00±0.91a 0.44±0.20c 0.45±0.20c 0.02±0.00d Shin Ichinose 4.96±0.07c 97.50±0.27a 0.64±0.26bc 0.66±0.03bc 0.03±0.00c Kenmochi 7.14±0.09a 97.60±0.25a 1.13±0.12a 1.15±0.12a 0.05±0.01b Mahalii 5.85±2.02b 96.99±0.72a 0.82±0.27b 0.85±0.28ab 0.07±0.00a CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RGR, relative growth rate. a,b,c,dMeans followed by different letters in the same columns are significantly different (LSD, P<0.01). Table 2. Nutritional indices of third instar larvae of Glyphodes pyloalis on different hosts (means±SE). Host CI AD ECI ECD RGR Ichinose 5.20±0.10c 94.64±1.20a 1.51±0.16a 1.54±0.07a 0.04±0.01b Shin Ichinose 4.83±0.60c 91.69±0.89b 1.32±0.12b 1.45±0.13b 0.17±0.02a Kenmochi 9.23±0.40a 95.12±0.55a 0.98±0.22c 1.05±0.24c 0.06±0.02b Mahalii 7.88±0.26b 94.60±0.50a 1.55±0.12a 1.64±0.13a 0.08 ±0.01b CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RGR, relative growth rate. a,b,cMeans followed by different letters in the same columns are significantly different (LSD, P<0.01). No n- co mm er cia l u se on ly d=3; P<0.0003) were on Mahalii and Ichinose (0.07±0.00 and 0.02±0.00, respectively) (Table 1). The highest (95.12±0.55%) and lowest (91.69±0.89%) AD values (F=10.51; df=3; P<0.0038) of the third instar larvae of G. pyloalis were on Kenmochi and Shin Ichinose, respectively. The Shin Ichinose and Ichinose showed the highest and lowest values of RGR (F=51.57; df=3; P<0.0001) (0.17±0.02 and 0.04±0.01), respectively. The highest (9.23±0.40%) and lowest (4.83±0.60%) CI values (F=100.63; df=3; P<0.0001) were on Kenmochi and Shin Ichinose, respectively (Table 2). In the fourth instar, the highest (95.23±0.74) and lowest (91.77±1.46) values of AD (F=6.71; df=3; P<0.0141) were on Kenmochi and Shin Ichinise. The larvae fed on Shin Ichinose had the highest ECI (F=9.58; df=3; P<0.005) and ECD (F=16.69; df=3; P<0.0001) values (0.80±0.06 and 0.97±0.21, respectively). However, the lowest value of ECI and ECD (0.40±0.03 and, 0.41±0. 03 respectively) was observed on Kenmochi (Table 3). It was observed that larvae fed on Kenmochi in the fifth instar had the highest CI (F=26.84; df=3; P<0.0002) and AD (F=0.35; df=3; P<0.7885) values (4.60±0.73 and 89.22±5.60, respectively) and the lar- vae that fed on Mahalii had the lowest values of CI and AD (1.86±0.06 and 77.54±5.63). The highest and lowest RGR values (F=29.67; df=3; P<0.0001) were observed on Mahalii and Kenmochi (0.10±0.00 and 0.04±0.10, respectively) (Table 4). In most cases, the highest and lowest values of AD (F=7.48; df=2, 116; P<0.01) were on the second and fifth instars, respectively. The highest and lowest values of ECI (F=8.75; df=2, 114; P<0.01) and ECD (F=15.56; df=2, 113; P<0.01) were on the fifth and fourth instars, respectively. Biochemical assessments The results of biochemical assessments of G. pyloalis larvae fed on different host are shown in Table 5. The larvae reared on Mahalii (0.97±0.02 U/mg) showed the highest levels of proteolytic activity. However, protease activity was the lowest in midgut extracts from larvae fed on Kenmochi (0.75±0.03 U/mg) (F=6.86, df=3, P<0.0013). It was observed that larvae fed on Mahalii had the highest activity of trypsin (F=26.8, df=3, P<0.0001) and elastase (F=58.86, df=3, P<0.008), (2.30±0.0 and 2.13±0.00 U/mg, respectively) and the larvae that fed on Kenmochi had the lowest activity of trypsin and elastase (0.54±0.00 and 1.36±0.02 U/mg, respectively). The larvae reared on Kenmochi (1.31±0.01 U/mg) showed the highest activity of chy- motrypsin (F=6.35, df=3, P<0.003). As for proteolytic activity, the highest levels of amylase activity (F=1.51, df=3, P<0.0028) were found on the larvae that fed on Mahalii (0.17±0.10 U/mg). As illustrated in Table 5, the highest activity level of ALT (F=59.27, df=3, P<0.0001) and AST (F=3.59, df=3, P<0.006) were found on Shin Ichinose (8.92±0.69 and 15.65±2.44 U/mg, respectively) and the lowest on Kenmochi. The larvae that fed on Mahalii had the highest ALP (F=3.55, df=3, P<0.006) and ACP (F=8.49, df=3, P<0.003) values (0.09±0.05 and 0.11±0.07 U/mg, respectively) and were lowest on Ichinose (0.02±0.01and 0.04±0.01, respectively). The highest total protein was observed on Mahalii, while the low- est was on Kenmochi (1.19±0.20, 0.89±0.12, respectively) (F=4.39, df=3, P<0.0019). It was observed that larvae fed on Mahalii had the highest tre- halose (F=3.43, df=3, P<0.003) and cholesterol (F=0.49, df=3, P<0.0009) values [21.14±2.53 (mg/dL) and 0.08±0.04 (mg/dL) respec- tively], and larvae reared on Shin Ichinose showed the highest level of glucose (F=10.45, df=3, P<0.003) 0.98±0.02 (mg/dL) (Table 5). Chemical analyses of the leaves The results of leaf analysis demonstrated that phosphorus con- tent was highest in Shin Ichinose and lowest in Ichinose (0.34±0.02 and 0.24±0.01), respectively. The other two varieties were not signif- icantly different from each other (F=6.57; df=3; P>0.0150). Potassium content was not significantly different among the varieties (F=2.76; df=3; P>0.1113), while the highest nitrogen content was observed in Mahalii (3.07±0.24) and the lowest in Kenmochi (2.07±0.68) (F=5.88; df=3; P>0.0202). Similarly, protein content was highest in Mahalii and lowest in Kenmochi (19.18±0.52 and 12.93±2.67, respectively). However, calcium content was highest in Shin Ichinose and Kenmochi and lowest in Mahalii and Ichinose (F=8.03; df=3; P>0.0085) (Table 6). [Journal of Entomological and Acarological Research 2014; 46:1633] [page 45] Article Table 3. Nutritional indices of fourth instar larvae of Glyphodes pyloalis on different hosts (means±SE). Host CI AD ECI ECD RGR Ichinose 2.60±0.22bc 92.53±0.59b 0.76±0.25a 0.79±0.21ab 0.03±0.04b Shin Ichinose 2.23±0.18c 91.77±1.46b 0.80±0.06a 0.97±0.21a 0.02±0.00b Kenmochi 4.62±0.10a 95.23±0.74a 0.40±0.03b 0.41±0.03b 0.02±0.01b Mahalii 2.93±0.05b 91.90±1.29b 0.58±0.33ab 0.63±0.37ab 0.06±0.02a CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RGR, relative growth rate. a,b,cMeans followed by different letters in the same columns are significantly different (LSD, P<0.01). Table 4. Nutritional indices of fifth instar larvae of Glyphodes pyloalis on different hosts (means±SE). Host CI AD ECI ECD RGR Ichinose 3.14±0.27b 81.80±3.89ab 2.64±0.13ab 2.82±0.24b 0.05±0.01bc Shin Ichinose 2.34±0.19bc 81.98±0.52ab 3.10±0.09a 3.82±0.16a 0.06±0.01b Kenmochi 4.60±0.73a 89.22±5.60a 2.23±0.36b 2.81±0.50b 0.04±0.10c Mahalii 1.86±0.06c 77.54±5.63b 2.68±0.15ab 3.20±0.29ab 0.10±0.00a CI, consumption index; AD, approximate digestibility; ECI, efficiency of conversion of ingested food; ECD, efficiency of conversion of digested food; RGR, relative growth rate. a,b,cMeans followed by different letters in the same columns are significantly different (LSD, P<0.01). No n- co mm er cia l u se on ly [page 46] [Journal of Entomological and Acarological Research 2014; 46:1633] Discussion and conclusions The use of resistant varieties is one of the core strategies of an integrated pest management program, and secondary substances of plants or allelochemicals play a major role in plant resistance to pests (Wilson & Huffaker, 1976). Differences in allelochemical concentra- tions between host plant varieties can affect an insect’s performance as a larva (Martin & Pulin, 2004). It is generally accepted that low dietary protein can cause an increase in the rate at which larvae feed (Rausher, 1981; Slansky, 1993); conversely, a high protein diet can reduce feeding rates (Mattson, 1980). In our study, the highest level of protein was recorded in leaves of the variety Mahalii. However, the lowest CI was recorded in fifth instar G. pyloalis larvae fed on Mahalii leaves. Also, the highest CI was observed on the variety Kenmochi, which had the lowest leaf pro- tein content. Hemati et al. (2011) recorded that H. armigera larvae had the highest CI values when they were fed on tomato leaves, for which Kotkar et al. (2009) reported very low protein content. Significant differences were found within the nutritional indices, especially ECI and ECD values, of G. pyloalis reared on different mul- berry varieties, suggesting that the varieties have different nutritional value. Among nutritional indices, ECI may vary with the digestibility of food and the proportional amount of the digestible portion of food that is converted to body mass and metabolized for energy needed for vital activity (Abdel-Rahman & Al-Mozini, 2007). ECI is the insect’s ability to utilize the food ingested for growth and development, and ECD is a measure of the efficiency of conversion of digested food into growth (Senthil-Nathan et al., 2005). Change in ECD also indicates the overall increase or decrease of the proportion of digested food metabolized for energy (Koul et al., 2004). For the fifth larval instars, the highest ECI and ECD values were on Shin Ichinose, suggesting that the larvae were more efficient at con- version of ingested and digested food to body biomass with a high increase in larval weight. Despite Kenmochi’s having the highest CI value, it also had the lowest values of ECI and ECD (Table 4), indicat- ing that larvae feeding on this host were less effective in converting ingested and digested food into biomass. It is well known that the degree of food utilization depends on the digestibility of food, and the efficiency with which digested food is converted into biomass (Batista Pereira et al., 2002). The reduction in dietary utilization suggests that reduction in nutritional values may be a result of both behavioural and physiological effects (Senthil-Nathan et al., 2005). Approximate digestibility (AD) and efficiency of conversion of digested food (ECD) are inversely related (Waldbauer, 1968; Scriber & Slansky, 1981) and this is demonstrated in larvae reared on Kenmochi, which had the highest rate of AD and lowest rate of ECD. Reduced RGR due to decreased ECD, despite an observed increase in AD, was noticed in larvae of 4th and 5th instar reared on the variety Kenmochi. Growth reduction in response to a new environment (host) has been previously shown in phytophagous insects (Grabstein & Scriber, 1982; Sheppard & Friedman, 1990; Lazarevic & Peric- Mataruga, 2003). Lepidopteran larvae fed on high-nutrient food have increased growth rates and a shorter developmental period than larvae fed on low- nutrient food (Hwang et al., 2008). Our results showed that RGR values Article Table 5. Comparison of biochemical compounds of Glyphodes pyloalis on four varieties of mulberry. Compounds Ichinose Shin Ichinose Kenmochi Mahalii a-amylase (U/mg) 0.11±0.01b 0.10±0.05b 0.10±0.05b 0.17±0.10a Ptotease (U/mg) 0.89±0.01a 0.80±0.04ab 0.75±0.03b 0.97±0.02a Trypsin (U/mg) 2.14±0.00b 1.91±0.01c 0.54 ±0.00d 2.30±0.02a Chymotrypsin (U/mg) 0.66±0.02b 0.83±0.01b 1.31±0.01a 0.65±0.01b Elastase (U/mg) 1.75±0.01b 1.86±0.03a 1.36±0.02c 2.13±0.00a Protein (mg/dL) 0.97±0.04ab 1.03±0.02ab 0.89±0.12b 1.19±0.20a Glucose (mg/dL) 0.80±0.01b 0.98±0.02a 0.92±0.08a 0.95±0.01a Cholesterol (mg/dL) 0.01±0.00b 0.02±0.01b 0.02±0.01b 0.08±0.04a Glycogen (mg/dL) 0.02±0.00a 0.02±0.00a 0.02±0.00a 0.02±0.00a Trehalose (mg/dL) 12.86±2.63c 18.45±0.66ab 17.53±5.57b 21.14±2.53a Alkalinaminoteransferase (IU/L) 6.60±0.66b 8.92±0.69a 2.99±0.47c 6.03±0.27b Aspartate aminoteransferase (IU/L) 13.35±2.57a 15.65±2.44a 3.99±0.58b 6.95±1.59b Alkaline phosphatase (IU/L) 0.04±0.01b 0.04±0.01b 0.05±0.02b 0.09±0.05a Acids phosphatase (IU/L) 0.02±0.01b 0.02±0.01b 0.04±0.01b 0.11±0.07a a,b,c,dMeans followed by different letters in the same columns are significantly different (LSD, P<0.01). Table 6. Chemical analyses of leaves of four different varieties of mulberry (means±SE; ingredient %D.M.). Host P K N Ca Protein Ichinose 0.24±0.01b 2.31±0.05a 2.62±0.11ab 2.59±1.14b 16.37±0.72ab Shin Ichinose 0.34±0.02a 2.44±0.02a 2.76±0.61ab 3.22±0.22a 17.25±1.60ab Kenmochi 0.32±0.01ab 2.42±0.04a 2.07±0.68b 3.16±0.17a 12.93±2.67b Mahalii 0.32±0.16ab 2.47±0.39a 3.07±0.24a 2.59±0.48b 19.18±0.52a a,bMeans followed by different letters in the same columns are significantly different (LSD, P<0.01). No n- co mm er cia l u se on ly were highest on Mahalii and lowest on Kenmochi, indicating that Kenmochi was a low-nutrient food for larvae, and a longer period of development was therefore needed by the immature stages. Conversely, the Mahalii plants were high-nutrient foods for larvae, and a shorter period of development was needed by the immature stages. The data on nutritional indices for the fourth and fifth instars of G. pyloalis are not consistent. This is because the nutritional require- ments of the insect change through development, and such differences typically result in changes in food consumption and feeding behavior (Barton Browme, 1995). Analysis of nutritional indices can lead to an understanding of the behavioural and physiological bases of insect response to host plants (Lazarevic & Peric-Mataruga, 2003). The lower fitness of G. pyloalis on some hosts may be due to the presence of some secondary phytochemicals in these hosts, or the absence of primary nutrients necessary for growth and development. To obtain more appli- cable information for G. pyloalis control, more attention should be devoted to studying the demographic parameters of this pest under lab- oratory and field conditions, as well as to investigate its nutritional indices on different varieties of mulberry under field conditions. Isolation and study of distinct proteases and amylases is of little aid in ascertaining the composition of the midgut or designing PI-based approaches for insect resistance, especially when the insect has the ability to modify midgut compounds within a single generation in order to deactivate the influence of PIs (Patankar et al., 2001; Vinokurov et al., 2007). Insects adjust to plant PIs by producing inhibitor-insensitive, inhibitor-resistant and inhibitor-declining proteinases in their midgut to compensate for the influence of transgenic or dietary PIs (Jongsma et al., 1995; Broadway, 1997; Michaud, 1997; Girard et al., 1998; Giri & Kachole, 1998). Therefore, determination of the midgut protease and amylase activities induced upon intake of PIs will be essential for selecting PIs or a mixture of PIs for expanding insect resistance (Patankar et al., 2001). In the current research, higher protease activ- ities in the Mahalii-fed larvae may have been due to the high protein content of the diet or to the response of the insect to the dietary PIs that partially inhibit midgut protease activity (Patankar et al., 2001). Additionally, hyperproduction of proteases in response to ingested PIs leads to an extra load on the insect for energy and essential amino acids, resulting in a retardation of insect growth (Broadway & Duffy, 1986). The highest trypsin- and elastase-like activities were also in Mahalii-fed larvae compared with the other varieties, and proteases and trypsin activity in the larvae reared on Kenmochi and Shin Ichinose were lowest. Lectins are carbohydrate-binding proteins dis- tributed in different species of plants (Etler, 1986; Ratanapo, 2005). They play different roles in plants; many of them have a direct inhibito- ry effect on some digestive enzymes of higher animals and insects, including a-amylases (Thompson & Gabon, 1986; Fish & Thompson, 1991), and esterases and proteases (Belzunces et al., 1994; Thompson et al., 1986). Ratanapo et al. (2005) reported two mulberry leaf lectins, MLL1 and MLL2, that have an inhibitory effect on trypsin-like alkaline proteases purified from the digestive fluid of the fifth larval instar of the silkworm, Bombyx mori. Anti-proteolytic effect of lectins on the digestive proteases might occur prior to silkworm digestion of food pro- tein. In this research, lower activity of protease and trypsin in Shin Ichinose than in Ichinose may indicate the existence of lectin/s in this variety. However, the larvae fed on Kenmochi exhibited the highest chymotrypsin activity compared with other varieties. The tentative answer to this phenomenon could be the over-expression of chy- motrypsin-like enzymes in response to the trypsin inhibitors in this variety. Trypsin and chymotrypsin occur as multiple isoforms in insects (Lam et al., 1999, 2000; Lopes et al., 2006; Sato et al., 2008). The highest levels of amylase activity were found on the larvae that fed on Mahalii. The reason could be that this host had the highest balance of carbohydrates. According to chemical analyses of leaves, we observed that the varieties Mahalii and Kenmochi had the highest and lowest levels of protein, respectively. Hemati et al. (2011) showed that the larvae reared on the tomato cultivar Dehghan had the highest level of proteolytic and amylolytic activity. The aminotransferases are enzymes that catalyze the reaction between an amino acid and a keto acid. This reaction removes the amino group from the amino acid, leaving a keto acid and converting it into an amino acid. These enzymes serve as a link between the carbo- hydrates and protein metabolism and are altered during various physi- ological processes (Etebari et al., 2004; Shekari et al., 2008). Our results show that Shin Ichinose and Kenmochi had the highest and lowest level of ALT and AST, respectively. The reason may be that Kenmochi had the lowest balance of protein. ACP and ALP are the hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids in alkaline and acidic conditions, both of which were highest in Mahalii. According to the results of chemical analyses of the leaves, Mahalii leaves indeed had the highest level of protein (Table 6). Glycogen is a polymer of several glucose residues in a branched chain storage form (Klowden, 2007; Lide, 1998); both glucose and glycogen were highest in the larvae that were reared on Shin Ichinose. In this research, we observed that the larvae that were reared on Mahalii and Kenmochi had the highest and lowest total protein, respec- tively, which corresponds with other results showing that these vari- eties have the highest and lowest protein contents. In this study, we found that the chemical compounds present in the host plant can play an important role in the feeding activity, digestive enzyme activity and chemical compounds stored in the plant when fed upon by pests. Based on our results, the variety Mahalii had the high- est nutritional indices and the highest activity of digestive enzymes to make it the best host for G. pyloalis. The variety Kenmochi had the low- est activity of digestive enzymes and nutritional indices, identifying it as an unsuitable host. The low activity of digestive enzymes and nutritional indices of G. pyloalis on Kenmochi could possibly indicate the presence of some enzyme inhibitors in this variety. 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