POTATO LEAF EXTRACT AND ITS
COMPONENT,
␣-SOLANINE,
EXERT SIMILAR IMPACTS ON
DEVELOPMENT AND OXIDATIVE
STRESS IN
Galleria mellonella
L.
Zbigniew Adamski
Electron and Confocal Microscope Laboratory, Faculty of Biology, Adam Mickiewicz University, Pozna ´n, Poland
Zbigniew Adamski, Pawel Marciniak,
and Kazimierz Ziemnicki
Department of Animal Physiology and Developmental Biology, Faculty of Biology, Adam Mickiewicz University, Pozna ´n, Poland
Ender B ¨uy ¨ukg ¨uzel
Department of Molecular Biology and Genetics, Faculty of Arts and Science, B¨ulent Ecevit University, Zonguldak, Turkey
Meltem Erdem
Ahmet Erdoðan Vocational School of Health Services, B¨ulent Ecevit University, Zonguldak, Turkey
Kemal B ¨uy ¨ukg ¨uzel
Department of Biology, Faculty of Arts and Science, B¨ulent Ecevit University, Zonguldak, Turkey
Emanuela Ventrella, Patrizia Falabella,
Massimo Cristallo, Rosanna Salvia,
and Sabino Aurelio Bufo
Dipartimento di Scienze, Universit`a degli Studi della Basilicata, Potenza, Italy
Laura Scrano
Department of European and Mediterranean Cultures, University of Basilicata, Matera, Italy
Correspondence to: Professor Patrizia Falabella, Dipartimento di Scienze, Universit`a degli Studi della Basilicata, Via dell’Ateneo Lucano 10, 85100 Potenza, Italy. E-mail: patrizia.falabella@unibas.it
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 87, No. 1, 26–39 (2014) Published online in Wiley Online Library (wileyonlinelibrary.com).
C
Plants synthesize a broad range of secondary metabolites that act as natural defenses against plant pathogens and herbivores. Among these, potato plants produce glycoalkaloids (GAs). In this study, we analyzed the effects of the dried extract of fresh potato leaves (EPL) on the biological parameters of the lepidopteran, Galleria mellonella (L.) and compared its activity to one of the main EPL components, the GAα-solanine. Wax moth larvae were reared from first instar on a diet supplemented with three concentrations of EPL orα-solanine. Both EPL and α-solanine affected survivorship, fecundity, and fertility of G. mellonella to approximately the same extent. We evaluated the effect of EPL andα-solanine on oxidative stress in midgut and fat body by measuring malondialdehyde (MDA) and protein carbonyl (PCO) contents, both biomarkers of oxidative damage. We evaluated glutathione S-transferase (GST) activity, a detoxifying enzyme acting in prevention of oxidative damage. EPL and α-solanine altered MDA and PCO concentrations and GST activity in fat body and midgut. We infer that the influence of EPL on G. mellonella is not enhanced by synergistic effects of the totality of potato leaf components compared toα-solanine alone.C 2014 Wiley Periodicals, Inc.
Keywords: potato leaf extract; glycoalkaloids; Galleria mellonella;
development; oxidative stress; GST
INTRODUCTION
Plants synthesize a variety of secondary metabolites, some of them allelochemicals that act as natural defenses against herbivores and phytopathogens (Wittstock and Gershenzon, 2002; Friedman, 2002, 2006; Alotaiba and Elsayed, 2007). Allelochemicals include a wide range of compounds that exert antifeedant or toxic activities deterring insect herbivores (Duffey and Stout, 1996). Several studies assessed the possible use of plant extracts or pure allelochemicals as an alternative approach to insect pest management (Koul and Walia, 2009; Nenaah, 2011a).
Besides the macroscopic lethal effects following exposure to plant extracts or pure allelochemicals, it is useful to consider the sublethal toxicity, and the cellular or molecular alterations due to these compounds. The degradation of ingested allelochemicals is often associated with increased production of reactive oxygen species (ROS) that damage insect tissues by oxidizing cell components (Pardini, 1995; Girotti, 1998; Bokov et al., 2004). In mice and Drosophila melanogaster reduced oxidative damage can extend life span (Bokov et al., 2004). Phytophagous insects possess a complex set of antioxidants and detoxifying enzymes to reduce the flux of oxidative radicals generated by allelochemicals (Aucoin et al., 1991; Krishnan and Senhal, 2006). In insects, glutathione S-transferases (GSTs) act in detoxification of xenobiotics, sometimes conferring insecticide resistance (Che-Mendoza et al., 2009). Insecticide exposure leads to induction of GST activity in many insect species (Yu, 2004; Che-Mendoza et al., 2009).
Plants of the Solanaceae family produce a mixture of secondary metabolites consist-ing of individual compounds of diverse chemical structure. Among them, glycoalkaloids (GAs) exert toxic and/or dysmetabolic activity (Herb et al., 1975). α-Chaconine and α-solanine are the most abundant GAs in commercial cultivars of potato plants (Fried-man, 2004). They are produced in all parts of the plant. However, leaves, unripe fruits,
and flowers have the highest GA concentrations (Friedman, 2006). Potato GAs make up a plant defense mechanism (Friedman, 2006; Adamski et al., 2009; Marciniak et al., 2010; B ¨uy ¨ukg ¨uzel et al., 2013), and although knowledge regarding their activities on insect phys-iology and mechanisms of action is still limited (Krishnan and Kodrik, 2006), crude plant extracts may exert a greater effect than the individual allelochemicals due to possible synergisms of multiple compounds (Nenaah, 2011a; Ventrella et al., 2014). This led us to pose the hypothesis that the dried, total extracts of fresh potato leaves (EPL) exert more deleterious impact on wax moth larvae than pureα-solanine, a major GA component of potato leaves. In this article, we report on the outcomes of experiments designed to test our hypothesis.
MATERIALS AND METHODS
Insect Culture
Greater wax moth larvae and pupae were collected from infected hives in apicultural areas around Zonguldak, Turkey, and the newly emerged adults were used to maintain the stock culture. The insects were reared in 1,000-ml glass jars with an artificial diet (Charri`ere and Imdorf, 1999) at 30± 1°C, 65 ± 5% relative humidity in constant darkness. The standard diet was composed of 420 g of wheat bran, 150 ml of filtered honey, 150 ml of glycerol, 20 g of ground old dark honeycomb and 30 ml of distilled water. Fifteen newly emerged adult females were placed in the jars and provided with a piece of old honeycomb for egg deposition and feeding of newly hatched larvae. The methods used to prepare and dispense diets in containers to obtain eggs and placement of larvae on diets are described (B ¨uy ¨ukg ¨uzel et al., 2010).
The work was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for animal experiments.
Chemicals
All reagents were analytical grade. Pure α-chaconine (ࣙ95%) and α-solanine (ࣙ95%), were purchased from LabService Analytica (Bologna, Italy). Phenylmethylsulphonyl fluoride (PMSF), dithiotreitol (DTT), β-nicotinamide adenine dinucleotide phos-phate (NADPH), butylated hydroxytoluene (BHT), bovine serum albumin (BSA), Folin-Ciocalteu reagent, ethylendiaminetetraacetic acid (EDTA), thiobarbituric acid (TBA), trichloroacetic acid (TCA), guanidine hydrochloride, 2,4-dinitrophenylhydrazine (DNPH), streptomycin sulphate, dipotassium hydrogen phosphate (K2HPO4), potassium chloride (KCl), glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), hydrogen per-oxide (H2O2), glycerol, ethanol, sodium chloride (NaCl), phenylthiourea (PTU) were purchased from Sigma-Aldrich (St. Louis, MO).
Extract Preparation From Potato Leaves
Fresh leaves were sampled from potato plants (Solanum tuberosum L. cv. Spunta, Solanaceae Juss.), immediately freeze-dried and stored at –20°C. The extraction was carried out by a slight modification of previously described method (Marciniak et al., 2010). Briefly, freeze-dried material was ground to a fine powder using a laboratory mill and an aqueous solution containing 1% acetic acid was added in the ratio 1:75 (v/w). The suspension was stirred for about 2 h and then centrifuged at 3,828 RCF, 4°C, for 30 min. The pellet was extracted again as just described. The two liquid extracts were combined and filtered
through single-use 0.22μm nylon filters (Whatman, Maidstone, UK) and the extract was injected into a LC/MS system. For determination of EPL bioactivity, the liquid phase was removed by lyophilization.
Characterization of EPL by LC/ESI-MS
LC/ESI-MS analysis was carried out in positive mode using a LCQ Classic quadrupole ion trap mass spectrometer QITMS (Thermo Finnigan, San Jose, CA) equipped with a binary pump and a solvent degasser (Spectra System P4000). The column was a Supelcosil LC-ABZ, amide-C16 (5μm, 250 × 4.6 mm) with a guard column of the same material (Supelco Inc., Bellefonte, PA; Cataldi et al., 2005). The concentrations of major GAs in the potato leaf extract were determined by using authentic GA pure standards (LabService Analytica, Bologna, Italy).
General Experimental Procedures
Dried EPL was directly incorporated into diets at concentrations of 0.05, 0.15, or 0.3 g in 100 g of diets.α-Solanine (Crystal form, 95%) was directly incorporated into diets at con-centrations of 0.13, 0.39, or 0.79 mg in 100 g of diets. These concon-centrations were selected on the bases of calculatedα-solanine content in the dietary EPL concentrations. Control larvae were reared on diets without extract or pureα-solanine. The EPL concentrations used in this study were based on our preliminary tests performed on a large number of lepidopteran pest insects (unreported data). Using standard laboratory rearing condi-tions, two series of experiments were carried out to examine the effects of EPL on the insects. In the first series, the effects of dried extract on survivorship, egg production (fecundity), and egg hatchability (fertility) were determined. In the second series, we determined the influence of dietary dried extract on lipid peroxidation and protein oxi-dation levels and on GST activity in midgut and fat body of last instar larvae (7thinstar). Last instar larvae were used in the biochemical analyses because our preliminary exper-iments demonstrated that some plant allelochemicals exerted their main effects mostly on the larval stages of the insect (Hyrˇsl et al., 2007; B ¨uy ¨ukg ¨uzel et al., 2010). The first and second series of experiments were conducted in parallel using pureα-solanine in place of EPL.
Survivorship
First instar larvae were reared until adult emergence on the artificial diets amended with given concentrations of EPL or pureα-solanine (controls reared on untreated diet). Survivorship of seventh instar larval, pupal, and adult stages was recorded. Surviving seventh instar larvae were counted and transferred into another jar lined with a filter paper for pupation and adult emergence. The filter paper was used to provide a dry surface for pupation. Numbers of pupae and adults were recorded for each replicate. Each experiment was replicated four times with 15 larvae per replicate.
Fecundity and Fertility
Neonate larvae were reared to the adult stage on the experimental and control diets. Pairs of experimental females and males were mated in vials for 24 h, then females were placed in 30-ml plastic cups with screened lids. Females were allowed to oviposit in cups
for the next 2 days. After this period, adults were removed from the cups and eggs were transferred into Petri dishes using a fine brush. Total number of eggs laid per female per day was recorded (fecundity). Egg counts were performed in the Petri dish on a black background to make the eggs more visible. Numbers of eggs hatched per female per day and their percentage were recorded (fertility). Each experiment was replicated four times with 10 females per replicate. Egg production and larval emergence were monitored continuously from the first oviposition day until experiments were stopped.
Biochemical Assays
Experimental and control first instar larvae were reared until 7th-instar (last instar, 100– 150 mg/larva) and used for determining the content of (i) the lipid peroxidation product, malondialdehyde (MDA), (ii) protein oxidation product, protein carbonyls (PCO), and (iii) detoxification enzyme GST activity. All larvae were of the same age. Each experiment with three concentrations of EPL and control was replicated four times with 20 larvae per replicate and each experiment with three pureα-solanine concentrations and control was replicated four times with 15 larvae per replicate.
Before dissection, larvae were chilled on ice for 5 min and surface sterilized in 95% ethanol. Larvae were longitudinally sectioned from the first pair of thoracic legs to the third pair of abdominal appendages using dissection scissors and the midguts were iso-lated with fine-tipped forceps under stereo-microscope (Olympus SZ61, Tokyo, Japan). Malpighian tubules and gut contents were removed. Midgut and fat body were sepa-rately collected into a chilled Eppendorf tube charged with cold homogenization buffer [1.15% w/v KCl, 25 mM K2HPO4, 5 mM ethylen-diaminetetraacetic acid (EDTA), 2 mM phenylmethylsulphonyl fluoride (PMSF), 2 mM dithiotreitol (DTT), pH 7.4] and stored at –80°C. A few crystals of phenylthiourea (PTU) were added to each sample to prevent melanization (Li et al., 2012). The cryotubes were kept at room temperature until the tissue began to thaw. Extracts of midgut and fat body were prepared in a homogenization buffer at 4°C using an ultrasonic homogenizer (Bandelin Sonoplus, HD2070, Berlin, Ger-many) at 50 W for 40–50 s and a subsequent centrifugation was carried out at 10,000 RCF, 4°C, for 10 min. The resulting cell-free extracts were collected for biochemical analysis. A dual beam spectrophotometer (Shimadzu 1700, UV/VIS Spectrophotometer, Kyoto, Japan) was used for all absorbance measurements.
Malondialdehyde (MDA) content was determined after incubation at 95°C with TBA (1% w/v) according to Jain and Levine (1995). MDA reacts with TBA to form a colored complex. Absorbances were measured at 532 nm and MDA content was expressed as nmol mg−1protein by using 1.56× 105M−1cm−1as extinction coefficient.
Protein carbonyl (PCO) content was assayed according to Levine et al. (1990) with some modifications (Hussein et al., 2006). PCO was quantified spectrophotometrically after reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) leading to the formation of a stable 2,4-dinitrophenylhydrazone. Absorbances were measured at 370 nm and results were expressed as nmol mg−1 protein by using 0.22× 105 M−1cm−1as the extinction coefficient. Protein concentrations were measured spectrophotometrically and quantified with a BSA standard curve (Lowry et al., 1951). Protein carbonyls values were corrected eliminating the contributions of interfering substances by subtracting the A370/mg protein measured without DNPH.
Glutathione S-transferase (GST) activity was assayed by measuring the formation of the GSH and 1-chloro-2,4-dinitrobenzene (CDNB) conjugate (Habig et al., 1974). Absorbance changes were recorded at 340 nm for 3 min. The specific activity of GST was expressed as
Table 1. Effects of Dried Extract from Fresh Potato Leaves (EPL) on Survival of G. mellonella EPL (g/100 g of diet) Survival to seventh nstar (%) (Mean± SE)a Survival to pupal stage (%) (Mean± SE)a Survival to adult stage (%) (Mean± SE)a
0.000b 84.99± 4.32a 78.33± 3.63a 73.32± 6.23a
0.05 59.99± 4.08b 54.99± 4.92b 49.99± 3.72b
0.15 61.66± 4.93b 48.33± 2.76b 41.65± 4.33b
0.3 48.32± 11.63b 41.66± 11.63b 33.32± 11.78b
aFour replicates with 15 larvae per replicate. Values followed by the same letter are not significantly different from each other, P> 0.05 (χ2test, LSD Test).
bControl (without EPL).
nmol GSH-CDNB conjugate formed min-1mg-1protein using an extinction coefficient of 9.6 mM−1cm−1. All obtained values were corrected eliminating the contributions of nonenzymatic reactions using corresponding substrate in phosphate buffer (50 mM, pH 7.0).
Statistical Analysis
Data on the egg production, hatchability, MDA and PCO content in midgut and fat body and GST activity were subjected to one-way analysis of variance (ANOVA). To determine significant differences between means, the least significant difference (LSD) test (User’s manual, version 10. SPSS, Chicago, 1997) was used and significance was set at P< 0.05. Data on survivorship were compared by a chi-squared test (Snedecor and Cochran, 1989). When the estimated F andχ2values exceeded the probability of 0.05, the differences were considered significant. The effect of EPL andα-solanine at corresponding concentrations for both biological and biochemical parameters were analyzed by the unpaired Student’s t-test and P valuesࣘ 0.05 were considered statistically significant.
The inhibitory effects of either tested extract orα-solanine survival, fecundity, fertility were estimated using probit analysis. The effect-concentration equations, their correlation coefficients and extract concentration values for 50 and 95% of inhibition (IC50and IC95, respectively) were obtained (Finney, 1971).
RESULTS
Characterization of EPL by LC/ESI-MS
The two major EPL GAs wereα-chaconine and α-solanine at 4.03 mg g−1and 2.62 mg g−1. We recorded minor GAs, dehydrochaconine, dehydrosolanine, and a hydroxy-derivative ofα-chaconine.
The Effects of EPL andα-Solanine on Survivorship of G. mellonella
As described in Table 1, diet containing EPL significantly reduced the 7th-instar larval, pupal, and adult survivorship, in comparison to controls, similar to the effect ofα-solanine (Table 2). There was a negative correlation between EPL andα-solanine concentrations and insect survival (Tables 3 and 4).
Table 2. Effects ofα-Solanine on Survival of G. mellonella α-Solanine (mg/100 g of diet) Survival to seventh instar (%) (Mean± SE)a Survival to pupal stage (%) (Mean± SE)a Survival to adult stage (%) (Mean± SE)a
0.000b 83.75± 2.07a 77.50± 2.80a 73.75± 2.07a
0.13 72.50± 1.25a 66.25± 2.72a 55.00± 1.77b
0.39 51.25± 2.07b 40.00± 1.77b 36.25± 2.07c
0.79 45.00± 3.06b 35.00± 1.77b 16.25± 2.07d
aFour replicates with 15 larvae per replicate. Values followed by the same letter are not significantly different from each other, P> 0.05 (χ2test, LSD Test).
bControl (withoutα-solanine).
Table 3. Correlation Between Dried Extract from Fresh Potato Leaves (EPL) Concentration and Measured Developmental Parameters Parameter Equation of IC curve Probit of inhibition Y= log A+ B × log × (log of concentration) Correlation factor IC50/95% confidence interval/(g/100 g of diet) IC95/95% confidence interval/(g/100 g of diet) Survival to seventh instar Y= 4.999 + 0.373 × log conc. –0.8409 0.10016× 100/0.721 × 10−1– 0.1391× 102/ 0.2584× 105/0.7812 × 10−2– 0.8547× 1011/ Survival to pupal stage Y= 5.188 + 0.564 × log conc –0.8553 0.4650× 10 0/0.1455 × 100– 0.1486× 101/ 0.3825× 103/0.5985 × 100– 0.2446× 106/ Survival to imagoes Y= 5.980 + 1.183 × log conc –0.8834 0.1483× 10 0/0.1111 × 100– 0.1979× 100/ 0.3650× 101/0.9671 × 100– 0.1378× 102/ Fecundity Y= 5.463 + 0.843 × log conc. –0.9341 0.2821× 10 0/0.1569 × 100– 0.5072× 100/ 0.2522× 102/0.9809 × 100– 0.6487× 103/ Fertility Y= 6.011 + 1.389 × log conc. –0.9643 0.1871× 100/0.1435 × 100– 0.2439× 100/ 0.2860× 101/0.9864 × 100– 0.8294× 101/
Table 4. Correlation Betweenα-Solanine Concentration and Measured Developmental Parameters
Parameter Equation of IC curve Probit of inhibition Y= log A+ B × log × (log of concentration) Correlation factor IC50/95% confidence interval/(g/100 g of diet) IC95/95% confidence interval/(g/100 g of diet) Survival to seventh instar Y= 5.6654 + 1.2938× log conc –0.9763 0.3064× 100/0.2073 × 100– 0.4517× 100/ 0.5719× 101/0. 1350 × 101– 0.2422× 102/ Survival to pupal stage Y= 6.0561 + 1.5751× log conc –0.9647 0. 2135× 100/0.1664 × 100– 0.2740× 100/ 0.2366× 101/0.9516 × 100– 0.5881× 101/ Survival to imagoes Y= 6.6545 + 1.8120 x log conc –0.9879 0.1221× 100/0.1008 × 100– 0. 1481× 100/ 0.9882× 101/0.5551 × 100– 0.1795× 101/ Fecundity Y= 5.4583 + 0.9180× log conc –0.9098 0.3168× 100/0.1806 × 100– 0. 5557× 100/ 0.1963× 102/0.1553 × 101– 0.2480× 103/ Fertility Y= 5.7659 + 3.8399× log conc –0.7940 0. 6317× 100/0.1953 × 100– 0.2043× 101/ 0.1694× 101/0.1143 × 100– 0.2512× 102/
Table 5. Effects of Dried Extract from Fresh Potato Leaves (EPL) on Fecundity and Fertility of G. mellonella EPL (g/100 g of diet) Fecundity (eggs/day/female) (Mean± SE)a Fertility (hatchability of eggs) (%) (Mean± SE)a
0.000b 67.46± 6.65a 69.88± 3.32a 0.05 52.21± 2.43ab 55.29± 4.01a 0.15 30.97± 9.55b 38.96± 9.15ab 0.3 24.00± 11.96b 27.24± 10.50b F 4.17 4.40 df 3 3 P 0.031 0.026
aFour replicates with 10 adults per replicate. Values followed by the same letter are not significantly different from each other, P> 0.05 (LSD Test).
bControl (without EPL).
Table 6. Effects ofα-Solanine on Fecundity and Fertility of G. mellonella α-solanine
(mg/100 g of diet)
Fecundity (eggs/day/female) (Mean± SE)a
Fertility (hatchability of eggs) (%) (Mean± SE)b 0.000b 67.35± 0.84a 69.55± 1.33a 0.13 49.75± 1.00b 50.36± 4.21b 0.39 46.50± 0.91b 33.86± 0.94c 0.79 30.65± 1.65c 20.01± 1.41d F 129.72 61.23 df 3 3 P <0.0001 <0.0001
aFour replicates with 10 adults per replicate. Values followed by the same letter are not significantly different from each other, P> 0.05 (LSD Test).
bControl (withoutα-solanine).
The Effects of EPL andα-Solanine on Fecundity and Fertility of G. mellonella
The effects of EPL and, separately,α-solanine on fecundity and fertility are presented in Tables 5 and 6. Relatively to controls, adults from larvae reared on diets with the highest concentrations of EPL orα-solanine significantly decreased both egg production and fertility. Fecundity or fertility was negatively correlated with EPL and α-solanine concentrations (Tables 3 and 4).
The Effects of EPL andα-Solanine on MDA, PCO Content, and GST Activity
Dietary EPL and α-solanine led to a significantly increased MDA content in fat body (Fig. 1A and B), and the highest EPL concentration decreased MDA content in midgut (Fig. 1A). Midgut MDA content was increased by allα-solanine concentrations (Fig. 1B). Dietary EPL and α-solanine altered PCO contents (Fig. 2A and B). Diets supple-mented with the highest EPL concentration significantly increased PCO content in lar-val midgut and fat body (Fig. 2A). Similarly, all the concentrations of pureα-solanine significantly increased PCO content (Fig. 2B) in larval midgut and fat body. Larvae reared on diet with the highest EPL concentration had increased midgut GST activity (Fig. 3A); EPL led to a significant decrease in fat body GST activity in a concentration-dependent manner (Fig. 3A). Similarly, diet supplemented with all the concentrations ofα-solanine
Figure 1. (A, B) Effects of dried leaf potato extract (a) and ofα-solanine (b) on midgut and fat body malon-dialdehyde (MDA) content of G. mellonella larvae. Each histogram bar represents the mean of four replicates± SE (a, n20 and b, n15) in each group. Vertical bars represent standard error. Means followed by the same letter are not statistically significantly different (P> 0.05, LSD test).
significantly increased midgut GST activity and led a significant decrease in GST enzymatic activity in fat body, in comparison to controls (Fig. 3B).
DISCUSSION
Our data show that EPL andα-solanine influence the biology of wax moth larvae, as seen in the red flour beetle Tribolium castaneum and the rice weevil Sitophilus oryzae (Nenaah, 2011a). However, in those cases the extract was more toxic than individual purified phy-tochemicals, indicating to us that the extract components acted in synergy. Potato GAs also reduced the growth rate, food consumption rate, and food utilization by adult Tro-goderma granarium, probably due to their toxic and antifeedant activity (Nenaah, 2001b). Solanaceous GAs generally reduce insect reproductive capacity. The tomato GA,
Figure 2. (A, B) Effects of dried leaf potato extract (a) and ofα-solanine (b) on midgut and fat body protein carbonyl (PCO) content of G. mellonella larvae. Each histogram bar represents the mean of four replicates± SE (a, n20 and b, n15) in each group. Vertical bars represent standard error. Means followed by the same letter are not statistically significantly different (P> 0.05, LSD test).
α-tomatine, was highly toxic to eggs of the diamondback moth (Plutella xylostella), re-ducing egg hatch from 90 to 20% (Friedman, 2002). Similarly,α-chaconine decreased reproduction rates of the potato aphid Macrosiphum euphorbiae (G ¨untner et al., 1997). α-solanine and α-chaconine added to an artificial diet, in concentrations lower or similar to those observed in potato leaves, reduced fecundity, feeding, and increased mortality in adults peach potato aphids Myzus persicae (Fragoyiannis et al., 1998).
Our results demonstrate that G. mellonella larvae reared on the diet with addition of EPL or α-solanine underwent decreased survivorship, fertility, and hatchability. In particular, development to adulthood was reduced by both treatments. Nonfeeding pupae do not accumulate additional toxins, however their metabolic processes are influenced by GAs accumulated during the larval stages. Lethal effects (IC50 and IC95) of pure α-solanine were obtained with lower concentrations, compared to EPL treatments. We infer
Figure 3. (A, B) Effects of dried leaf potato extract (a) andα-solanine (b) on midgut and fat body glutathione S-transferase (GST) activity of G. mellonella larvae. Each histogram bar represents the mean of four replicates± SE (a, n20 and b, n15) in each group. Vertical bars represent standard error. Means followed by the same letter are not statistically significantly different (P> 0.05, LSD test).
that, contrary to our hypothesis, potential synergies among whole-leaf compounds did not lead to increased negative impacts on the experimental insects, compared toα-solanine. Our data showed that EPL andα-solanine influenced MDA, PCO contents, as well as GST activity. The midgut is generally susceptible to oxidative injury by ingested xenobi-otics. The fat body acts in detoxification, because xenobiotic molecules and their metabo-lites are generally transferred along with nutrients from midgut to fat body (Arrese and Soulages, 2010). EPL andα-solanine increased the fat body MDA and PCO contents and decreased GST enzymatic activity. We speculate that this is the consequence of disrupting the normal homeostatic pro-oxidant/anti-oxidant activities during exposure to the xeno-biotics contained in EPL (Charri`ere and Imdorf, 1999). Based on the effects reported for all the concentrations ofα-solanine on fat body MDA and PCO contents as well as GST activity, we infer that α-solanine alters these biochemical parameters at least in the fat
body. In midguts exposed to the highest concentration of EPL, the enhancement of GST activity in correlation to reduced MDA and relatively elevated PCO content suggested to us that GST has a protective role against EPL-induced oxidative stress. The increased GST activity could be a midgut response to the rise of oxidative radicals (Sheehan et al., 2001) and could represent a way of preparing the insect for adaptive metabolic response to the elevation of lipid peroxidation (Ahmad et al., 1989). Our interpretation is that the oxidative stress in midgut was limited by increased GST activity, as suggested for some other insect groups (Leszzynski et al., 1994). We observed increased midgut MDA content in larvae treated with all the concentrations ofα-solanine, from which we infer that the pure GA exerted stronger oxidative activity than the EPL.
ACKNOWLEDGMENTS
We are grateful to Dr. Gennaro Sansone for reading and providing useful comments on a draft of this report, and Dr. Rita Armentano for the English language editing. We also thank the Interdepartmental Center for Scientific Equipment (CIGAS) of University of Basilicata, Potenza, for LC/ESI-MS instrumental availability.
The authors declare that there is no conflict of interests regarding the publication of this article.
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