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Geranium intoxication induces detoxification enzymes in the Japanese beetle, Popillia japonica Newman
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Geranium intoxication induces detoxification enzymes in the Japanese beetle, Popillia japonica Newman Adekunle W. Adesanyaa,b,⁎, David W. Helda,⁎, Nannan Liua a b
Department of Entomology and Plant Pathology, Auburn University, 301 Funchess Hall, Auburn, AL 36830, United States Department of Entomology, Washington State University, 279A FSHN building, Pullman, WA 99163, United States
A R T I C L E I N F O
A B S T R A C T
Keywords: Quisqualic acid P450 activity GST activity Carboxylesterase activity Allelochemical toxicity Biopesticide
Popillia japonica is a generalist herbivore that feeds on > 300 host plant species in at least 72 plant families. It is unknown why P. japonica, despite possessing active detoxification enzymes in its gut, is paralyzed when feeding on the petals of one of its preferred host plant, Pelargonium × hortorum, or on artificial diet containing quisqualic acid (QA), the active compound in zonal geranium. We hypothesized that Pelargonium × hortorum or QA do not induce activity of the cytochrome P450, glutathione S transferase (GST), and carboxylesterase (CoE) detoxification enzymes in P. japonica. In this study, P. japonica were fed petals of zonal geranium or agar plugs containing QA, or rose petals, another preferred but non-toxic host. Midgut enzyme activities of P450, GST, and CoE were then assayed after 6, 12, or 24 h of feeding. In most cases, P450, GST, and CoE activities were significantly induced in P. japonica midguts by geranium petals and QA, though the induction was slower than with rose petals. Induced enzyme activity reached a peak at 24 h after consumption, which coincides with the period of highest recovery from geranium and QA paralysis. This study shows that toxic geranium and QA induce detoxification enzyme activity, but the induced enzymes do not effectively protect P. japonica from paralysis by QA. Further investigation is required through in vitro studies to know if the enzymes induced by geranium are capable of metabolizing QA. This study highlights a rare physiological mismatch between the detoxification tool kit of a generalist and its preferred host.
1. Introduction Plants possess an arsenal of chemical defenses in the form of organic and inorganic compounds, i.e. allelochemicals, which can negatively affect non-adapted herbivores [1–3] with significant biological consequence such as reduced growth, fecundity, or survival [4]. In fact, the entomotoxicity of some signature plant allelochemicals (e.g. pyrethrins, azadirachtin) have been exploited as pesticides to control insects. On the other hand, insect herbivores have coevolved with plant allelochemicals by developing survival strategies such as sequestration of plant toxins [5,6], increased rates of excretion, feeding on less defended plant parts, or the use of detoxification enzymes for biochemical metabolism to overcome the hurdle of plant toxins present in their diets [7,8]. Detoxification enzymes are well documented among insect herbivores as the primary biochemical adaptation to allow utilization of plants that would otherwise be toxic, and is also a major mechanism for insecticide resistance [3,8–12]. Acute toxicity can occur when an insect herbivore consumes a plant with novel or toxic secondary plant compound(s) [3]. Some herbivores
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may experience a temporary bout of torpidity followed by recovery. Specialist and generalist caterpillars forced to consume latex of Asclepias curassavica containing toxic cardenolides experienced spasms and immobility for up to 3 d. Upon recovery, these larvae were able to continue to develop successfully on their host [13]. The Japanese beetle (Popillia japonica) is another dietary generalist that consumes leaves, flowers and fruits of > 300 host plants of agricultural, horticultural and ornamental importance [14]. However, one host plant of P. japonica, zonal geranium (Pelargonium × hortorum L. G. Bailey) is known to induce torpidity or paralysis of adults when flower petals are consumed [15,16]. Paralysis is often fatal in the field, where intoxicated beetles suffer direct mortality or are consumed by predators [15]. Under laboratory conditions, P. japonica will recover from this effect in 24 h or less, and repeatedly prefer to consume geranium flowers even in the presence of other non-toxic hosts such as linden (Tilia spp.) [17]. Quisqualic acid (QA), an agonist of glutamate receptors in muscles (neurotoxin), is the active compound in geranium petals responsible for paralysis in Japanese beetles [18]. The insecticidal activity of geranium plants and its extracts are well documented against different taxa of
Corresponding authors at: Department of Entomology and Plant Pathology, Auburn University, 301 Funchess Hall, Auburn, AL 36830, United States. E-mail addresses: [email protected] (A.W. Adesanya), [email protected] (D.W. Held).
http://dx.doi.org/10.1016/j.pestbp.2017.07.008 Received 20 May 2017; Received in revised form 25 July 2017; Accepted 28 July 2017 0048-3575/ © 2017 Published by Elsevier Inc.
Please cite this article as: Adesanya, A.W., Pesticide Biochemistry and Physiology (2017), http://dx.doi.org/10.1016/j.pestbp.2017.07.008
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insects: flies [19], cockroaches [20], ants [21] and termites [21]. However very little is known about the physiological response of insects to geranium ingestion. The phenomenon of torpidity/paralysis preceding recovery suggests that immobility may be a period when physiological detoxification mechanisms are being induced to minimize the potential effects of the ingested toxin [22], i.e. metabolic detoxification of xenobiotics. Although the exact mechanism allowing recovery from paralysis in P. japonica is unknown, the activation/induction of detoxification enzymes used by the beetles to defend against the plethora of allelochemicals in their diet has been hypothesized [17]. P. japonica possess inducible detoxification enzymes for metabolizing host plant allelochemicals [23,24] when feeding on a diversity of host plants. These detoxification enzymes include cytochrome P450s (P450s), glutathione S transferases (GSTs), and carboxylesterases (CoEs). The induction of these suites of enzymes is significantly influenced by the suitability of the host plants [23]. Despite the apparent effectiveness of its detoxification enzymes against most allelochemicals, P. japonica is still severely paralyzed by consuming just one geranium petal with a relatively low quantity (~ 4.9 ng) of QA [18], suggesting that QA intoxication may either cause a delayed detoxification induction, inhibit enzyme activity, or act on the nervous system before an induction of the detoxification enzymes can occur. Inhibition of detoxification enzymes should keep levels similar to beetles consuming non-toxic hosts or perhaps non-feeding individuals. A delay in induction may explain beetles' recovery from the toxicity of QA, especially in the lab as documented by Potter and Held [17]. During this time, isoform(s) of detoxification enzymes that can effectively metabolize this unique toxin can be induced. Also, in Manduca sexta larvae experiencing an induced intoxication upon nicotine ingestion have an increase in P450 activity correlated with recovery and continued nicotine consumption [22]. A similar mechanism may play a role in the recovery of P. japonica from geranium-induced paralysis. This study was conducted to understand the involvement of detoxification enzymes (P450s, GSTs and CoEs) in the paralysis of adult Japanese beetles by geranium and QA. The paralysis of P. japonica resulting from consumption of geranium petals or QA may interfere with the induction of P450, GST, and CoE detoxification enzymes in the midgut. Understanding the connection between the P. japonica paralysis by QA and activities of detoxification enzymes will shed light on how a generalist herbivore is seemingly defenseless against a toxin in an otherwise preferred host plant.
potting mix (Earthgro, Marysville, OH). Plants were potted in March 2014 and maintained in a greenhouse (25–27 °C, 14 h L: 10 h D) at Auburn University through July 2014. Plants were watered daily and fertilized twice during the growing period with lo-start liquid fertilizer (15-9-12, eveRRiS: Dublin, OH). No pesticides were applied in the greenhouse while the plants were being maintained. Petals of Rosa × radazz ‘Knockout’ (Rosaceae) (herein referenced as rose) were freshly collected from a planting of approximately 20 rose plants on the Auburn University campus. Rose petal is a palatable, nontoxic and preferred host that boosts P. japonica fitness and survival [25,26]. Petals (geranium and rose) used in the feeding experiments were freshly harvested < 6 h before each experiment. To ensure representative samples, each petal was plucked from different plants in each planting. 2.4. Enzyme activity of P. japonica's midgut in response to geranium This experiment compared the midgut activity of detoxification enzymes (P450, GST and CoE) of adult P. japonica after consumption of either zonal geranium petals or rose petals. Beetles were provided freshly harvested uninfested rose or geranium petals and allowed to feed for 6 h, 12 h or 24 h. As an additional control to establish the baseline of enzyme titer at each time period, we held another set of beetles in similar conditions but without food for the same time periods. A previous study showed that starving beetles within this duration had no significant effect on the activities of P450s, GSTs and CoEs [7]. All beetles were individually housed in 37 ml sterile waxed-bottom transparent plastic cups at 25–27 °C with a 14:10 L:D photoperiod. Moistened filter paper was placed at the bottom of each cup to prevent desiccation and provide humidity. Each beetle received either two rose or two geranium petals pinned to the center of the cup [16]. The mass of petal tissue (geranium or rose) consumed by each beetle was calculated as the difference in weight before and after feeding, using a balance and digital imaging software (Image J, http://imageJ.net) to estimate the area of petal consumed, as described in Adesanya et al. [23]. To account for petal desiccation, the area of petals consumed was converted to equivalent fresh weight using the measured mass/area ratio. Prior observations showed no significant desiccation or weight loss of petals within 24 h when held under the described experimental conditions. The number of beetles paralyzed by geranium consumption at each time period was scored. After feeding, a beetle was considered paralyzed when it could not move or extend its legs and was unable right itself when placed on its back [16–18]. Each treatment group (i.e. host plant × time) had 15–20 beetles in a randomized complete block experimental design with four replicates. Since the beetles were field collected, each block was based on the time beetles were collected from the field in order to reduce variation. The midgut tissue of the beetles was removed by dissection and later assayed for enzyme activity.
2. Materials and methods 2.1. Reagents and supplies Except where mentioned, all reagents and supplies used in this study were purchased from VWR Scientific.
2.5. Enzyme activity of P. japonica in response to feeding on QA This experiment evaluated midgut enzyme activity and paralysis of adult P. japonica associated with feeding on agar plugs containing toxic but non-deterrent doses of QA. In a preliminary test, we determined that 1.0 μg of QA per agar plug was sufficient to induce paralysis yet not significantly deter feeding in adult P. japonica. This concentration of QA was used in the subsequent experiments. We administered QA to beetles using an artificial diet of solid agar plugs [18,27]. Each agar plug (diameter × height: 0.5 × 1.1 cm) contained 10 μl of 0.1 M sucrose, a common phagostimulant used in Japanese beetle feeding assays [27]. Controls in this experiment were agar plugs containing sucrose but with no QA. To ensure that a uniform amount of QA was dispersed in the agar plug, a syringe was used to inject QA into individual agar plugs before they were fully solidified. Feeding assays were conducted with beetles held individually in petri dishes at 25–27 °C with 14:10 L:D photoperiod. Agar plugs (with or without QA) were individually weighed before and after feeding and
2.2. Source of insects Adult P. japonica were collected at Town Creek Park in Auburn AL (32.5978° N, 85.4808° W) using traps (Pherocon® JB trap, Trécé, Adair, OK) baited with a food lure (2-phenyl-ethyl propionate, eugenol, and geraniol; 3:7:3 ratio) in the summer months of 2014. There were no planted zonal geraniums at this location. The beetles were observed behaviorally in the lab to be active and void of any morphological defect before use in the feeding assays. Female beetles were held without food for 4–6 h after field collection prior to being used in the feeding assays. 2.3. Source and preparation of plant material Zonal geranium (Pelargonium × hortorum ‘Patriot Bright Red’) plants were transplanted into black plastic pots filled with sterilized 2
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The amount of 7-hydroxycoumarin produced was measured using a microplate reader (Cytation 3: Biotek) at a wavelength of 390 nm and 465 nm for excitation and emission respectively. The reaction between NADPH and 7EC in the absence of microsomal fraction was the control reaction. Serial dilutions of 100 μM of 7-hydroxycoumarin under the above conditions were used to generate a reference standard curve. P450 activity was expressed as pmoles of 7-hydroxycoumarin formed per min per mg protein.
the difference (in mg) was used to determine the amount consumed. Unused agar plugs were left under similar conditions as a control to account for weight loss; however weight loss was found to be insignificant over the duration of this experiment. The incidence of paralysis, amount of food ingested and the activities of detoxification enzymes in beetles' guts were determined at 12 h and 24 h. We did not test feeding duration at 6 h because there were no differences in consumption between 6 and 12 h. Non-feeding beetles were removed from the data set before analysis. Each treatment group had 15–20 beetles and was replicated independently three times.
2.6.4. GST activity assay GST activity of cytosolic fractions was determined by the ability to catalyze the conjugation of glutathione (GSH) and chloro-2,4-dintrobenzene (CDNB), as previously described Habig et al. [32]. Each reaction well (transparent flat-bottom 96-well plate) contained freshly prepared 2.5 mM GSH, 0.1 mg of the enzyme source (cytosolic fraction), and phosphate buffer added to a final volume of 200 μl. Reactions were incubated for 3 min at 25 °C, after which 10 μl of 30 mM CDNB (freshly dissolved in alcohol) was added. The control reaction was GSH and CDNB under the same conditions in the absence of enzyme. Absorbance at 340 nm was measured for 5 min using a plate reader in kinetic mode (Cytation 3: Biotek, Winooski, VT, USA). GST activity was expressed as micromoles CDNB conjugated per min per mg protein using the extinction co-efficient of 5.3 mM− 1 cm− 1 for S-(2,4-dinitrophenyl) glutathione.
2.6. Dissection of midguts After feeding, beetles were pinned on an ice-chilled waxed-bottom petri dish covered with Parafilm to prevent contamination, and dissected under a binocular microscope. Beetles' guts were excised using sterilized dissecting scissors and forceps. The guts from beetles with similar diet and feeding periods were pooled together and immediately transferred into 1.5 ml Eppendorf tubes placed on dry ice. Food residue in the midguts was removed by washing the guts in ice cold 0.1 M phosphate buffer (pH 7.5) before being stored temporarily in a −80 °C freezer. 2.6.1. Extraction of microsomes and cytosol We used a method similar to that described by Adesanya et al. [23] and Lee and Scott [28] to extract microsomes and cytosol. Beetle guts were homogenized in 10 ml of ice-cold homogenization buffer (0.1 M phosphate buffer pH 7.5, fortified with 10% glycerol, 1 mM EDTA, 0.1 mM DTT, 1 mM PMSF and 1 mM PTU). The homogenate was filtered through double-layered cheesecloth and the filtrate was centrifuged at 10,000g for 30 min at 4 °C (Beckman Allegra 25R equipped with a type TA-14-50 rotor). The pellet (nuclear debris and other materials) was discarded and the supernatant was transferred into another centrifuge tube and spun in an ultra-centrifuge (Sorvall, Discovery 90SE) at 100,000g for 1 h at 4 °C. The resulting supernatant (cytosolic fraction) was removed and used as the source of enzymes for the GST and CoE activity assays. Pellets (microsomes), the source of P450 enzyme, were dissolved in a re-suspension buffer (0.1 M phosphate buffer pH 7.5, 20% glycerol, 1 mM EDTA, 0.1 mM DTT and 1 mM PMSF). All the steps described were performed on ice. The microsomes and cytosol were temporarily stored at − 80 °C.
2.6.5. CoE activity assay Esterase activity was determined based on the hydrolysis of αnaphthylacetate (α-NA) to α-naphthol, previously described by van Asperen [33]. Each reaction well contained 60 μl of phosphate buffer (0.04 M, pH 7.0), 0.1 mg of the enzyme source (cytosolic fraction) and 80 μL of 0.3 mM of α-NA solution. The reaction was incubated at 37 °C for 30 min and terminated by the addition of 20 μL stop solution (two parts of 1% Fast Blue BB salt and five parts of 5% sodium dodecyl sulfate). Color change was allowed to develop for 15 min at 25 °C, and absorbance was measured at 600 nm (hydrolysis of α-NA) using a microplate reader (Cytation 3: Bio-Tek®, Winooski, VT, USA). A reference standard curve was generated by measuring absorbance at 600 nm of serial dilutions of α-naphthol dissolved in acetone and under similar condition to the enzymatic reactions.
2.6.2. Enzyme assays 2.6.2.1. Protein content determination:. Protein concentration in each sample (microsomes or cytosol) was determined using the Bradford method [29] with Bio-Rad Dye reagent. Serial dilutions of bovine serum albumin were used to generate a reference standard curve. There were three technical replicate measurements of protein from each biological sample using a UV-VIS spectrophotometer (Du 640, Beckman Coulter, Brea, CA).
Two-way ANOVA was used to test whether feeding durations and diet have significant effect on beetle's consumption (mg/h) and the enzymatic activity of P450s, GSTs and CoEs. LSD post hoc tests were used to identify significant differences among the treatment means. Logistic regression analysis was used to test the effect of feeding duration on the incidence of paralysis caused by geranium or QA intoxication, using the χ2 statistic. All analyses were done using JMP (SAS JMP version Pro 11, SAS Corp.).
2.6.3. P450 activity assay P450 activity was assayed according to the ability of microsomal protein from beetles' guts to convert 7-ethoxycoumarin (a model substrate) to 7-hydroxycoumarin by O-deethylation reaction. The method used was modified from earlier studies by Lee and Scott [30] and Guo et al. [31]. An aliquot of the microsomal fraction, 40 μl of ECOD buffer (50 mM Tris-base, 150 mM KCl and 1 mM EDTA, pH 7.8) and 4 nM of 7-ethoxycoumarin (7-EC) was added to each well of a 96-well plate (black flat-bottom BD Falcon). Freshly prepared 0.1 μM NADPH was added to each reaction well to initiate the reaction. Reaction mixtures were incubated for 30 min at 30 °C. The auto-fluorescence of NADPH was reduced by adding 0.3 μM oxidized glutathione and 0.5 units of glutathione reductase (Sigma–Aldrich, St. Louis, MO) to each well. The reaction was stopped after 10 min at 25 °C by adding 120 μl of stop solution (50% acetonitrile and 50% 0.05 M, pH 10 Trizma-base buffer).
3. Results
2.7. Data analyses
3.1. Feeding activity, paralysis and detoxification enzyme activity of P. japonica in response to geranium P. japonica consistently consumed more mass of rose petals than zonal geranium at each tested feeding duration (6, 12 and 24 h) (Fig. 1) showing that geranium intoxication reduces P. japonica's feeding activity (F(1, 18) = 389, P < 0.0001). Beetles' rate of consumption of rose (mg/h) was 4-fold higher than geranium at 6 h, 5.2-fold at 12 h and 3fold at 24 h (F(1, 18) = 60.1, P < 0.0001). When examining geranium consumption over time, beetles consumed significantly lower amounts of geranium at 12 h than at 6 h or 24 h, for which there was no difference in the rate of geranium consumption (Fig. 1, F(2, 15) = 24.4, P < 0.0001). 3
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point in this study, whereas rose induced 7.7 and 6-fold increases in GST activity at 6 h and 12 h of feeding respectively, relative to starved beetles, but not there was no difference at 24 h (Fig. 3B, F(2, 45) = 42.7, P < 0.0001). There was no change in starvation-induced COE activity over time. Consumption of geranium petals did not induce CoE until 24 h of feeding, while, consumption of rose petals induced a higher CoE response at 6 h and 12 h feeding duration but not at 24 h, indicating a quicker enzymatic response to rose relative to geranium (Fig. 3C, F(2, 63) = 3.4, P = 0.037). 3.2. Feeding activity, paralysis and detoxification enzyme activity of P. japonica in response to QA consumption The addition of QA to agar plugs significantly reduced the consumption rate of beetles by 38.7% and 13.7% at 12 h and 24 h respectively (F(1, 8) = 11.6, P < 0.001); Fig. 4). At 12 h, 86% of beetles were paralyzed from feeding on QA compared to 50% paralysis at 24 h, reflecting a significant recovery from intoxication (χ2 = 13.4 P = 0.0019) (Fig. 5). Feeding on QA-treated agar plugs generally induced activities of all three enzyme groups relative to agar only (Fig. 6). Consumption of agar plugs without QA (control) did not induce activity of any of the three enzymes at 24 h relative to 12 h (Fig. 6: A, B and C). Feeding on QA induced greater P450 and GST activity at 24 h relative to 12 h (Fig. 6A and B respectively). CoE activity declined at 24 h from a 2-fold induction at 12 h (Fig. 6C, F(1, 20) = 17.5, P < 0.0001). At 24 h, QA induced a 1.5-fold increase in P450 activity (F(1, 20) = 13.5, P = 0.0015) and 1.7-fold increase in GST activity (F(1, 20) = 7.9, P = 0.01).
Fig. 1. The effect of diet (rose, geranium) and feed duration (6, 12, 24 h) on adult Japanese beetle feeding. Diet (F(1, 18) = 389, P < 0.0001), Time (F(1, 18) = 60.1, P < 0.0001), Diet × Time (F(1, 18) = 24.4, P < 0.0001). The bars on each treatment represent standard error of each mean. Letters on x axis denote treatment received by beetles: G and R represent the diet treatment of either geranium or rose respectively. The corresponding numbers with each represent the duration of the exposure to the diet treatment (6, 12, or 24 h).
4. Discussion P. japonica is a generalist herbivore that encounters a diverse array of potentially toxic phytochemicals while foraging on numerous host plants. Detoxification enzymes (P450s, GSTs and CoEs) are used by Japanese beetles to allow polyphagy [23,24]. Although these enzymes successfully metabolize defensive compounds from many host plants, consumption of zonal geranium petals delivers a dose of QA sufficient to induce a temporal paralysis that can reduce longevity and survival in Japanese beetle. Potter and Held [17] speculated that phytochemicals in geranium petals may somehow circumvent the detoxification enzymes in the digestive system of P. japonica, thereby causing toxicity/ paralysis. This current study provides evidence that consumption of zonal geranium flower petals or QA in agar, although intoxicating, also induces enzyme activities in the midgut of Japanese beetles. However, the induction of these enzymes does not completely protect the beetles from geranium intoxication, as evidenced by the high percentage of beetles (> 50%) that remain paralyzed even after 24 h of feeding on geranium petals or QA treated agar plugs. QA-induced paralysis in adult Japanese beetles is temporary in the laboratory but often fatal in the field [16,17]. Recovery from geranium/ QA-induced paralysis by Japanese beetles varied with time. A 1.0 μg dose of QA in an agar plug with 0.1 M sucrose was sufficient to paralyze 86% of the tested beetles (at 12 h), which was similar to what we observed for beetles feeding on geranium petals (87% at 12 h). However, at 24 h feeding duration, there was a significant reduction in the numbers of beetles paralyzed by geranium petals (59%) and QA (50%), i.e. beetle recovery. It is most likely that the enzymatic response at 24 h confers some resistance to geranium/QA intoxication, which is also reflected in the increased consumption rate of geranium petals or QA agar plugs by the beetles at 24 h. Potter and Held [17] also noted that Japanese beetles were able to consume a greater petal mass of zonal geranium upon recovery from geranium-induced torpidity. Between these periods (12 h and 24 h), geranium feeding resulted in upregulation of P450 and CoE activity while QA-feeding resulting in an increase in P450 and GST activity and a decline in CoE activity. P450, GST and CoE enzymes are likely involved in the recovery of the beetles from QA
Fig. 2. The effect of feeding duration on the paralysis of P. japonica by geranium petal, χ2 = 10.78 P = 0.0048.
There was a significant effect of feeding duration on the number of beetles that were paralyzed by geranium petals (χ2 = 10.78 P = 0.0048). The highest incidence of paralysis (87%) was observed in beetles that fed on geranium for 12 h. The number of beetles paralyzed at 6 h (70%) and 24 h (59%) were similar (P = 0.27) despite the rate of consumption of geranium petals increasing significantly between 12 h and 24 h. More than 50% of beetles that consumed geranium petals were paralyzed at each time point (6, 12 or 24 h) (Fig. 2). Consumption of geranium and rose petals induced activity of one or more enzyme groups (P450s, GSTs and CoEs) relative to non-feeding, but the magnitude of the enzyme induction varied by time and plant species i.e. significant interaction between diet and time for: P450 (F(4, 63) = 11.6, P < 0.0001), GST (F(4, 63) = 8.3, P < 0.0001) and CoE (F(4, 63) = 15.7, P < 0.0001). Feeding-induced enzyme activity was evident in beetles that consumed rose (P450, GST and CoE) or geranium petals (P450 and CoE) relative to the non-feeding control beetles. Generally, consumption of rose petals elicited different enzyme responses compared to consumption of zonal geranium petals. Rose induced higher enzyme activity relative to geranium at 6 and 12 h of feeding except for GST (Fig. 3A, B and C). Starvation did not induce P450 activity across time, while geranium induced significant P450 activity (2.3, 2 and 2.2-fold increases at 6, 12 and 24 h respectively) compared to starved beetles. Rose induced significant P450 activity (3 and 2-fold) at 6 h and 12 h but not at 24 h (Fig. 3A, F(2, 63) = 43, P < 0.0001). GST activity in starved beetles remained stable between 6 h and 12 h of feeding but significantly increased at 24 h. Interestingly, geranium did not induce significant GST activity at any measured time 4
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Fig. 3. A: P450 activity in P. japonica that consumed rose and geranium petals at different time periods. Diet (F(2, 63) = 43, P < 0.0001), Time (F(2, 63) = 6.5, P = 0.0026), Diet × Time (F(4, 63) = 11.6, P < 0.0001). B: GST activity in P. japonica that consumed rose and geranium petals at different time periods. Diet (F(2, P < 0.0001) Time (F(2, 45) = 1.6, P = 0.27) 45) = 42.7, Diet × Time (F(4, 45) = 8.3, P < 0.0001). C: CoE activity in P. japonica that consumed rose and geranium petals at different time periods. Diet (F(2, 63) = 3.4, P = 0.037), Time (F(2, 63) = 11.8, P < 0.0001), Diet × Time (F(5, 63) = 15.7, P < 0.0001). The bars on each treatment represent standard error of each mean. Letters on x axis denote treatment received by beetles: S, G and R represent the diet treatment of either no food, geranium or rose respectively. The corresponding numbers with each represent the duration of the exposure to the diet treatment (6, 12, or 24 h).
Fig. 5. The effect of feeding duration on the paralysis of P. japonica by quisqualic acid, χ2 = 13.4 P = 0.0019.
Fig. 4. Average mass (mg) of agar plugs with/without QA consumed by P. japonica at different time periods. Diet (F(1, 8) = 187, P < 0.0001), Time (F(1, 8) = 644, P < 0.0001), Diet × Time (F(1, 8) = 11.6, P < 0.001). The bars on each treatment represent standard error of each mean. Letters on x axis denote treatment received by beetles: A12-agar for 12 h, Q12-agar + QA for 12 h, A24-agar for 24 h, Q24-agar + QA for 24 h.
The optimal dose of QA that induced paralysis of Japanese beetle in this study (1.0 μg) is ten-fold higher compared to that of a previous study (0.1 μg) [18]. However, differences in experimental condition and design could be responsible for this variation. Aside from any cofounding factor, this suggests that different populations of Japanese beetles might vary in tolerance/susceptibility to QA. This form of phenotypic plasticity is typical of most naturally occurring arthropod populations to natural and synthetic pesticides [12,37]. Japanese beetle is an invasive pest in North America that gained entry in via the port of New Jersey in early nineteenth century [14]. Hence, it is likely that a century of geographic range expansion could lead to genetic variation resulting in phenotypic plasticity toward a naturally occurring but unique toxin like QA. The observed variation in enzyme (P450, GST and CoE) induction patterns by geranium and QA reflects differences in enzymes responses between the synthetic form of QA and natural forms in planta. Other phytochemicals present in planta (e.g. flavonoids, geraniol) may compete as substrates for detoxification enzymes in the midgut of P.
intoxication. This is because the activities of these enzymes generally peaked at 24 h after feeding on QA, while the incidence of paralysis significantly reduced. Previous studies [12,34,35] have also implicated detoxification enzymes in reducing the effects of toxic allelochemicals on insect herbivores. Transcriptomic studies [36] in other herbivoreplant systems have revealed feeding on novel or toxic host plants usually triggers a suite of detoxification enzymes, but very few or a single isoenzyme is actually responsible for circumventing the host plant toxin. Future in vitro studies will be required to understand the direct interaction of QA and the gut enzymes of Japanese beetles, and will provide further insight into which of these enzymes play vital roles in the metabolism of QA. Furthermore, analysis of the transcriptome of Japanese beetles will help identify the isoforms of detoxification enzymes that are involved in the response to geranium/QA intoxication.
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Fig. 6. A: P450 activity in P. japonica that consumed agar plugs with/without QA at different feeding duration. Diet (F(1,20) = 31, P < 0.09), Time (F(1, 20) = 13.5, P = 0.0015), Diet × Time (F(1, 20) = 1.18, P < 0.29). B: GST activity in P. japonica that consumed agar plugs with/without QA at different time periods. Diet (F(1, P = 0.045) Time (F(1, 20) = 18.3, P = 0.0004), 20) = 4.6, Diet × Time (F(1, 20) = 7.9, P = 0.01). C: CoE activity in P. japonica that consumed agar plugs with/without quisqualic at different time periods. Diet (F(1, 20) = 238, P < 0.0001) Time (F(1, 20) = 41, P = 0.0026), Diet × Time (F(1, 20) = 17.5, P < 0.0001). Letters on x axis denote treatment received by beetles: A12-agar for 12 h, Q12agar + QA for 12 h, A24-agar for 24 h, Q24-agar + QA for 24 h.
However, enzyme induction does not completely prevent toxicity of QA/zonal geranium indicating a mismatch between Pelargonium × hortorum chemistry and P. japonica's detoxification kit. Evolutionarily, it is not surprising that toxins in Pelargonium are novel to P. japonica since they share no natural history. Pelargonium spp. originate from South Africa [39] and the beetle, originating from Asia, would have experience with members of Geraniaceae but not Pelargonium spp. Future studies in this plant-insect interaction would benefit from comparisons between non-toxic plants in the Geraniaceae such as P. × scarboroviae [16] and the toxic hybrids of P. × hortorum.
japonica or inhibit detoxification protein induction. Hence, allelochemicals other than QA present in geranium petals possibly have an effect on detoxification enzymes or stress responses. Neal and Berenbaum [35] reported that gut enzymes from Papilio polyxenes were sensitive to phytosynergists like myristicin and safrole, present in umbellifers, which increases the toxicity of in planta furanocoumarins relative to synthetic forms. Francis et al. [38] also observed significant variation in the GST activity in aphids that were fed on Brassica plants and aphids that fed on allelochemicals typical of Brassicaceae (i.e. glucosinolate) incorporated into artificial diet. Further investigation is required to identify other phytochemicals present in geranium that could be acting as synergists for the toxicity of QA to P. japonica. Our experiment with agar plugs provides insight into enzymatic responses to QA intoxication independent of other phytochemicals. This experimental design allows for indirect comparison of enzyme activity from consumption in planta versus artificial doses of QA. We found that CoE activity was induced coincident with significant paralysis when beetles consumed agar plugs with QA (i.e. at 12 h). CoE activity also increased over time when beetles consumed geranium petals and was greatest at 24 h when paralysis decreased. The increase in P450 and GST activities are coincident with a decrease in paralysis at 24 h in the agar plug experiment, suggesting that induction of P450, GTS and CoE are all involved in P. japonica's physiological response to QA intoxication. Snyder and Glendinning [22] reported a similar response in tobacco budworms feeding on toxic nicotine, where increases in P450 activity coincided with increases in nicotine consumption and reduction in intoxication.
Acknowledgements Young's Plant Farm, Auburn AL, generously donated the geraniums used in this study. This work was supported by the Alabama Agricultural Experiment Station through Hatch funding from the National Institute of Food and Agriculture, USDA. Thanks to Amy Worthington, Nate Hardy, Paul Nabity and Fang Zhu for reviewing the earlier drafts of this manuscript and Ting Li, Youhui Gong and Emily Wine for technical support. References [1] G.S. Fraenkel, The raison d'etre of secondary plant substances, Science 129 (1959) 1466–1470. [2] R.I. Krieger, P.P. Feeny, C.F. Wilkinson, Detoxication enzymes in the guts of caterpillars: an evolutionary answer to plant defenses? Science 172 (1971) 579–581. [3] L.M. Schoonhoven, J.J. Van Loon, M. Dicke, Insect-Plant Biology, Oxford University Press, 2005 (on Demand). [4] J.A. Fordyce, A.A. Agrawal, The role of plant trichomes and caterpillar group size on growth and defence of the pipevine swallowtail Battus philenor, J. Anim. Ecol. 70 (2001) 997–1005. [5] S. Malcolm, L. Brower, Evolutionary and ecological implications of cardenolide sequestration in the monarch butterfly, Cell. Mol. Life Sci. 45 (1989) 284–295.
5. Conclusion The present study shows that QA in its pure form or in planta elicits induction of detoxification enzymes in the gut of adult P. japonica. 6
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Geranium intoxication induces detoxification enzymes in the Japanese beetle, Popillia japonica Newman Adekunle W. Adesanyaa,b,⁎, David W. Helda,⁎, Nannan Liua a b
Department of Entomology and Plant Pathology, Auburn University, 301 Funchess Hall, Auburn, AL 36830, United States Department of Entomology, Washington State University, 279A FSHN building, Pullman, WA 99163, United States
A R T I C L E I N F O
A B S T R A C T
Keywords: Quisqualic acid P450 activity GST activity Carboxylesterase activity Allelochemical toxicity Biopesticide
Popillia japonica is a generalist herbivore that feeds on > 300 host plant species in at least 72 plant families. It is unknown why P. japonica, despite possessing active detoxification enzymes in its gut, is paralyzed when feeding on the petals of one of its preferred host plant, Pelargonium × hortorum, or on artificial diet containing quisqualic acid (QA), the active compound in zonal geranium. We hypothesized that Pelargonium × hortorum or QA do not induce activity of the cytochrome P450, glutathione S transferase (GST), and carboxylesterase (CoE) detoxification enzymes in P. japonica. In this study, P. japonica were fed petals of zonal geranium or agar plugs containing QA, or rose petals, another preferred but non-toxic host. Midgut enzyme activities of P450, GST, and CoE were then assayed after 6, 12, or 24 h of feeding. In most cases, P450, GST, and CoE activities were significantly induced in P. japonica midguts by geranium petals and QA, though the induction was slower than with rose petals. Induced enzyme activity reached a peak at 24 h after consumption, which coincides with the period of highest recovery from geranium and QA paralysis. This study shows that toxic geranium and QA induce detoxification enzyme activity, but the induced enzymes do not effectively protect P. japonica from paralysis by QA. Further investigation is required through in vitro studies to know if the enzymes induced by geranium are capable of metabolizing QA. This study highlights a rare physiological mismatch between the detoxification tool kit of a generalist and its preferred host.
1. Introduction Plants possess an arsenal of chemical defenses in the form of organic and inorganic compounds, i.e. allelochemicals, which can negatively affect non-adapted herbivores [1–3] with significant biological consequence such as reduced growth, fecundity, or survival [4]. In fact, the entomotoxicity of some signature plant allelochemicals (e.g. pyrethrins, azadirachtin) have been exploited as pesticides to control insects. On the other hand, insect herbivores have coevolved with plant allelochemicals by developing survival strategies such as sequestration of plant toxins [5,6], increased rates of excretion, feeding on less defended plant parts, or the use of detoxification enzymes for biochemical metabolism to overcome the hurdle of plant toxins present in their diets [7,8]. Detoxification enzymes are well documented among insect herbivores as the primary biochemical adaptation to allow utilization of plants that would otherwise be toxic, and is also a major mechanism for insecticide resistance [3,8–12]. Acute toxicity can occur when an insect herbivore consumes a plant with novel or toxic secondary plant compound(s) [3]. Some herbivores
⁎
may experience a temporary bout of torpidity followed by recovery. Specialist and generalist caterpillars forced to consume latex of Asclepias curassavica containing toxic cardenolides experienced spasms and immobility for up to 3 d. Upon recovery, these larvae were able to continue to develop successfully on their host [13]. The Japanese beetle (Popillia japonica) is another dietary generalist that consumes leaves, flowers and fruits of > 300 host plants of agricultural, horticultural and ornamental importance [14]. However, one host plant of P. japonica, zonal geranium (Pelargonium × hortorum L. G. Bailey) is known to induce torpidity or paralysis of adults when flower petals are consumed [15,16]. Paralysis is often fatal in the field, where intoxicated beetles suffer direct mortality or are consumed by predators [15]. Under laboratory conditions, P. japonica will recover from this effect in 24 h or less, and repeatedly prefer to consume geranium flowers even in the presence of other non-toxic hosts such as linden (Tilia spp.) [17]. Quisqualic acid (QA), an agonist of glutamate receptors in muscles (neurotoxin), is the active compound in geranium petals responsible for paralysis in Japanese beetles [18]. The insecticidal activity of geranium plants and its extracts are well documented against different taxa of
Corresponding authors at: Department of Entomology and Plant Pathology, Auburn University, 301 Funchess Hall, Auburn, AL 36830, United States. E-mail addresses: [email protected] (A.W. Adesanya), [email protected] (D.W. Held).
http://dx.doi.org/10.1016/j.pestbp.2017.07.008 Received 20 May 2017; Received in revised form 25 July 2017; Accepted 28 July 2017 0048-3575/ © 2017 Published by Elsevier Inc.
Please cite this article as: Adesanya, A.W., Pesticide Biochemistry and Physiology (2017), http://dx.doi.org/10.1016/j.pestbp.2017.07.008
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insects: flies [19], cockroaches [20], ants [21] and termites [21]. However very little is known about the physiological response of insects to geranium ingestion. The phenomenon of torpidity/paralysis preceding recovery suggests that immobility may be a period when physiological detoxification mechanisms are being induced to minimize the potential effects of the ingested toxin [22], i.e. metabolic detoxification of xenobiotics. Although the exact mechanism allowing recovery from paralysis in P. japonica is unknown, the activation/induction of detoxification enzymes used by the beetles to defend against the plethora of allelochemicals in their diet has been hypothesized [17]. P. japonica possess inducible detoxification enzymes for metabolizing host plant allelochemicals [23,24] when feeding on a diversity of host plants. These detoxification enzymes include cytochrome P450s (P450s), glutathione S transferases (GSTs), and carboxylesterases (CoEs). The induction of these suites of enzymes is significantly influenced by the suitability of the host plants [23]. Despite the apparent effectiveness of its detoxification enzymes against most allelochemicals, P. japonica is still severely paralyzed by consuming just one geranium petal with a relatively low quantity (~ 4.9 ng) of QA [18], suggesting that QA intoxication may either cause a delayed detoxification induction, inhibit enzyme activity, or act on the nervous system before an induction of the detoxification enzymes can occur. Inhibition of detoxification enzymes should keep levels similar to beetles consuming non-toxic hosts or perhaps non-feeding individuals. A delay in induction may explain beetles' recovery from the toxicity of QA, especially in the lab as documented by Potter and Held [17]. During this time, isoform(s) of detoxification enzymes that can effectively metabolize this unique toxin can be induced. Also, in Manduca sexta larvae experiencing an induced intoxication upon nicotine ingestion have an increase in P450 activity correlated with recovery and continued nicotine consumption [22]. A similar mechanism may play a role in the recovery of P. japonica from geranium-induced paralysis. This study was conducted to understand the involvement of detoxification enzymes (P450s, GSTs and CoEs) in the paralysis of adult Japanese beetles by geranium and QA. The paralysis of P. japonica resulting from consumption of geranium petals or QA may interfere with the induction of P450, GST, and CoE detoxification enzymes in the midgut. Understanding the connection between the P. japonica paralysis by QA and activities of detoxification enzymes will shed light on how a generalist herbivore is seemingly defenseless against a toxin in an otherwise preferred host plant.
potting mix (Earthgro, Marysville, OH). Plants were potted in March 2014 and maintained in a greenhouse (25–27 °C, 14 h L: 10 h D) at Auburn University through July 2014. Plants were watered daily and fertilized twice during the growing period with lo-start liquid fertilizer (15-9-12, eveRRiS: Dublin, OH). No pesticides were applied in the greenhouse while the plants were being maintained. Petals of Rosa × radazz ‘Knockout’ (Rosaceae) (herein referenced as rose) were freshly collected from a planting of approximately 20 rose plants on the Auburn University campus. Rose petal is a palatable, nontoxic and preferred host that boosts P. japonica fitness and survival [25,26]. Petals (geranium and rose) used in the feeding experiments were freshly harvested < 6 h before each experiment. To ensure representative samples, each petal was plucked from different plants in each planting. 2.4. Enzyme activity of P. japonica's midgut in response to geranium This experiment compared the midgut activity of detoxification enzymes (P450, GST and CoE) of adult P. japonica after consumption of either zonal geranium petals or rose petals. Beetles were provided freshly harvested uninfested rose or geranium petals and allowed to feed for 6 h, 12 h or 24 h. As an additional control to establish the baseline of enzyme titer at each time period, we held another set of beetles in similar conditions but without food for the same time periods. A previous study showed that starving beetles within this duration had no significant effect on the activities of P450s, GSTs and CoEs [7]. All beetles were individually housed in 37 ml sterile waxed-bottom transparent plastic cups at 25–27 °C with a 14:10 L:D photoperiod. Moistened filter paper was placed at the bottom of each cup to prevent desiccation and provide humidity. Each beetle received either two rose or two geranium petals pinned to the center of the cup [16]. The mass of petal tissue (geranium or rose) consumed by each beetle was calculated as the difference in weight before and after feeding, using a balance and digital imaging software (Image J, http://imageJ.net) to estimate the area of petal consumed, as described in Adesanya et al. [23]. To account for petal desiccation, the area of petals consumed was converted to equivalent fresh weight using the measured mass/area ratio. Prior observations showed no significant desiccation or weight loss of petals within 24 h when held under the described experimental conditions. The number of beetles paralyzed by geranium consumption at each time period was scored. After feeding, a beetle was considered paralyzed when it could not move or extend its legs and was unable right itself when placed on its back [16–18]. Each treatment group (i.e. host plant × time) had 15–20 beetles in a randomized complete block experimental design with four replicates. Since the beetles were field collected, each block was based on the time beetles were collected from the field in order to reduce variation. The midgut tissue of the beetles was removed by dissection and later assayed for enzyme activity.
2. Materials and methods 2.1. Reagents and supplies Except where mentioned, all reagents and supplies used in this study were purchased from VWR Scientific.
2.5. Enzyme activity of P. japonica in response to feeding on QA This experiment evaluated midgut enzyme activity and paralysis of adult P. japonica associated with feeding on agar plugs containing toxic but non-deterrent doses of QA. In a preliminary test, we determined that 1.0 μg of QA per agar plug was sufficient to induce paralysis yet not significantly deter feeding in adult P. japonica. This concentration of QA was used in the subsequent experiments. We administered QA to beetles using an artificial diet of solid agar plugs [18,27]. Each agar plug (diameter × height: 0.5 × 1.1 cm) contained 10 μl of 0.1 M sucrose, a common phagostimulant used in Japanese beetle feeding assays [27]. Controls in this experiment were agar plugs containing sucrose but with no QA. To ensure that a uniform amount of QA was dispersed in the agar plug, a syringe was used to inject QA into individual agar plugs before they were fully solidified. Feeding assays were conducted with beetles held individually in petri dishes at 25–27 °C with 14:10 L:D photoperiod. Agar plugs (with or without QA) were individually weighed before and after feeding and
2.2. Source of insects Adult P. japonica were collected at Town Creek Park in Auburn AL (32.5978° N, 85.4808° W) using traps (Pherocon® JB trap, Trécé, Adair, OK) baited with a food lure (2-phenyl-ethyl propionate, eugenol, and geraniol; 3:7:3 ratio) in the summer months of 2014. There were no planted zonal geraniums at this location. The beetles were observed behaviorally in the lab to be active and void of any morphological defect before use in the feeding assays. Female beetles were held without food for 4–6 h after field collection prior to being used in the feeding assays. 2.3. Source and preparation of plant material Zonal geranium (Pelargonium × hortorum ‘Patriot Bright Red’) plants were transplanted into black plastic pots filled with sterilized 2
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The amount of 7-hydroxycoumarin produced was measured using a microplate reader (Cytation 3: Biotek) at a wavelength of 390 nm and 465 nm for excitation and emission respectively. The reaction between NADPH and 7EC in the absence of microsomal fraction was the control reaction. Serial dilutions of 100 μM of 7-hydroxycoumarin under the above conditions were used to generate a reference standard curve. P450 activity was expressed as pmoles of 7-hydroxycoumarin formed per min per mg protein.
the difference (in mg) was used to determine the amount consumed. Unused agar plugs were left under similar conditions as a control to account for weight loss; however weight loss was found to be insignificant over the duration of this experiment. The incidence of paralysis, amount of food ingested and the activities of detoxification enzymes in beetles' guts were determined at 12 h and 24 h. We did not test feeding duration at 6 h because there were no differences in consumption between 6 and 12 h. Non-feeding beetles were removed from the data set before analysis. Each treatment group had 15–20 beetles and was replicated independently three times.
2.6.4. GST activity assay GST activity of cytosolic fractions was determined by the ability to catalyze the conjugation of glutathione (GSH) and chloro-2,4-dintrobenzene (CDNB), as previously described Habig et al. [32]. Each reaction well (transparent flat-bottom 96-well plate) contained freshly prepared 2.5 mM GSH, 0.1 mg of the enzyme source (cytosolic fraction), and phosphate buffer added to a final volume of 200 μl. Reactions were incubated for 3 min at 25 °C, after which 10 μl of 30 mM CDNB (freshly dissolved in alcohol) was added. The control reaction was GSH and CDNB under the same conditions in the absence of enzyme. Absorbance at 340 nm was measured for 5 min using a plate reader in kinetic mode (Cytation 3: Biotek, Winooski, VT, USA). GST activity was expressed as micromoles CDNB conjugated per min per mg protein using the extinction co-efficient of 5.3 mM− 1 cm− 1 for S-(2,4-dinitrophenyl) glutathione.
2.6. Dissection of midguts After feeding, beetles were pinned on an ice-chilled waxed-bottom petri dish covered with Parafilm to prevent contamination, and dissected under a binocular microscope. Beetles' guts were excised using sterilized dissecting scissors and forceps. The guts from beetles with similar diet and feeding periods were pooled together and immediately transferred into 1.5 ml Eppendorf tubes placed on dry ice. Food residue in the midguts was removed by washing the guts in ice cold 0.1 M phosphate buffer (pH 7.5) before being stored temporarily in a −80 °C freezer. 2.6.1. Extraction of microsomes and cytosol We used a method similar to that described by Adesanya et al. [23] and Lee and Scott [28] to extract microsomes and cytosol. Beetle guts were homogenized in 10 ml of ice-cold homogenization buffer (0.1 M phosphate buffer pH 7.5, fortified with 10% glycerol, 1 mM EDTA, 0.1 mM DTT, 1 mM PMSF and 1 mM PTU). The homogenate was filtered through double-layered cheesecloth and the filtrate was centrifuged at 10,000g for 30 min at 4 °C (Beckman Allegra 25R equipped with a type TA-14-50 rotor). The pellet (nuclear debris and other materials) was discarded and the supernatant was transferred into another centrifuge tube and spun in an ultra-centrifuge (Sorvall, Discovery 90SE) at 100,000g for 1 h at 4 °C. The resulting supernatant (cytosolic fraction) was removed and used as the source of enzymes for the GST and CoE activity assays. Pellets (microsomes), the source of P450 enzyme, were dissolved in a re-suspension buffer (0.1 M phosphate buffer pH 7.5, 20% glycerol, 1 mM EDTA, 0.1 mM DTT and 1 mM PMSF). All the steps described were performed on ice. The microsomes and cytosol were temporarily stored at − 80 °C.
2.6.5. CoE activity assay Esterase activity was determined based on the hydrolysis of αnaphthylacetate (α-NA) to α-naphthol, previously described by van Asperen [33]. Each reaction well contained 60 μl of phosphate buffer (0.04 M, pH 7.0), 0.1 mg of the enzyme source (cytosolic fraction) and 80 μL of 0.3 mM of α-NA solution. The reaction was incubated at 37 °C for 30 min and terminated by the addition of 20 μL stop solution (two parts of 1% Fast Blue BB salt and five parts of 5% sodium dodecyl sulfate). Color change was allowed to develop for 15 min at 25 °C, and absorbance was measured at 600 nm (hydrolysis of α-NA) using a microplate reader (Cytation 3: Bio-Tek®, Winooski, VT, USA). A reference standard curve was generated by measuring absorbance at 600 nm of serial dilutions of α-naphthol dissolved in acetone and under similar condition to the enzymatic reactions.
2.6.2. Enzyme assays 2.6.2.1. Protein content determination:. Protein concentration in each sample (microsomes or cytosol) was determined using the Bradford method [29] with Bio-Rad Dye reagent. Serial dilutions of bovine serum albumin were used to generate a reference standard curve. There were three technical replicate measurements of protein from each biological sample using a UV-VIS spectrophotometer (Du 640, Beckman Coulter, Brea, CA).
Two-way ANOVA was used to test whether feeding durations and diet have significant effect on beetle's consumption (mg/h) and the enzymatic activity of P450s, GSTs and CoEs. LSD post hoc tests were used to identify significant differences among the treatment means. Logistic regression analysis was used to test the effect of feeding duration on the incidence of paralysis caused by geranium or QA intoxication, using the χ2 statistic. All analyses were done using JMP (SAS JMP version Pro 11, SAS Corp.).
2.6.3. P450 activity assay P450 activity was assayed according to the ability of microsomal protein from beetles' guts to convert 7-ethoxycoumarin (a model substrate) to 7-hydroxycoumarin by O-deethylation reaction. The method used was modified from earlier studies by Lee and Scott [30] and Guo et al. [31]. An aliquot of the microsomal fraction, 40 μl of ECOD buffer (50 mM Tris-base, 150 mM KCl and 1 mM EDTA, pH 7.8) and 4 nM of 7-ethoxycoumarin (7-EC) was added to each well of a 96-well plate (black flat-bottom BD Falcon). Freshly prepared 0.1 μM NADPH was added to each reaction well to initiate the reaction. Reaction mixtures were incubated for 30 min at 30 °C. The auto-fluorescence of NADPH was reduced by adding 0.3 μM oxidized glutathione and 0.5 units of glutathione reductase (Sigma–Aldrich, St. Louis, MO) to each well. The reaction was stopped after 10 min at 25 °C by adding 120 μl of stop solution (50% acetonitrile and 50% 0.05 M, pH 10 Trizma-base buffer).
3. Results
2.7. Data analyses
3.1. Feeding activity, paralysis and detoxification enzyme activity of P. japonica in response to geranium P. japonica consistently consumed more mass of rose petals than zonal geranium at each tested feeding duration (6, 12 and 24 h) (Fig. 1) showing that geranium intoxication reduces P. japonica's feeding activity (F(1, 18) = 389, P < 0.0001). Beetles' rate of consumption of rose (mg/h) was 4-fold higher than geranium at 6 h, 5.2-fold at 12 h and 3fold at 24 h (F(1, 18) = 60.1, P < 0.0001). When examining geranium consumption over time, beetles consumed significantly lower amounts of geranium at 12 h than at 6 h or 24 h, for which there was no difference in the rate of geranium consumption (Fig. 1, F(2, 15) = 24.4, P < 0.0001). 3
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point in this study, whereas rose induced 7.7 and 6-fold increases in GST activity at 6 h and 12 h of feeding respectively, relative to starved beetles, but not there was no difference at 24 h (Fig. 3B, F(2, 45) = 42.7, P < 0.0001). There was no change in starvation-induced COE activity over time. Consumption of geranium petals did not induce CoE until 24 h of feeding, while, consumption of rose petals induced a higher CoE response at 6 h and 12 h feeding duration but not at 24 h, indicating a quicker enzymatic response to rose relative to geranium (Fig. 3C, F(2, 63) = 3.4, P = 0.037). 3.2. Feeding activity, paralysis and detoxification enzyme activity of P. japonica in response to QA consumption The addition of QA to agar plugs significantly reduced the consumption rate of beetles by 38.7% and 13.7% at 12 h and 24 h respectively (F(1, 8) = 11.6, P < 0.001); Fig. 4). At 12 h, 86% of beetles were paralyzed from feeding on QA compared to 50% paralysis at 24 h, reflecting a significant recovery from intoxication (χ2 = 13.4 P = 0.0019) (Fig. 5). Feeding on QA-treated agar plugs generally induced activities of all three enzyme groups relative to agar only (Fig. 6). Consumption of agar plugs without QA (control) did not induce activity of any of the three enzymes at 24 h relative to 12 h (Fig. 6: A, B and C). Feeding on QA induced greater P450 and GST activity at 24 h relative to 12 h (Fig. 6A and B respectively). CoE activity declined at 24 h from a 2-fold induction at 12 h (Fig. 6C, F(1, 20) = 17.5, P < 0.0001). At 24 h, QA induced a 1.5-fold increase in P450 activity (F(1, 20) = 13.5, P = 0.0015) and 1.7-fold increase in GST activity (F(1, 20) = 7.9, P = 0.01).
Fig. 1. The effect of diet (rose, geranium) and feed duration (6, 12, 24 h) on adult Japanese beetle feeding. Diet (F(1, 18) = 389, P < 0.0001), Time (F(1, 18) = 60.1, P < 0.0001), Diet × Time (F(1, 18) = 24.4, P < 0.0001). The bars on each treatment represent standard error of each mean. Letters on x axis denote treatment received by beetles: G and R represent the diet treatment of either geranium or rose respectively. The corresponding numbers with each represent the duration of the exposure to the diet treatment (6, 12, or 24 h).
4. Discussion P. japonica is a generalist herbivore that encounters a diverse array of potentially toxic phytochemicals while foraging on numerous host plants. Detoxification enzymes (P450s, GSTs and CoEs) are used by Japanese beetles to allow polyphagy [23,24]. Although these enzymes successfully metabolize defensive compounds from many host plants, consumption of zonal geranium petals delivers a dose of QA sufficient to induce a temporal paralysis that can reduce longevity and survival in Japanese beetle. Potter and Held [17] speculated that phytochemicals in geranium petals may somehow circumvent the detoxification enzymes in the digestive system of P. japonica, thereby causing toxicity/ paralysis. This current study provides evidence that consumption of zonal geranium flower petals or QA in agar, although intoxicating, also induces enzyme activities in the midgut of Japanese beetles. However, the induction of these enzymes does not completely protect the beetles from geranium intoxication, as evidenced by the high percentage of beetles (> 50%) that remain paralyzed even after 24 h of feeding on geranium petals or QA treated agar plugs. QA-induced paralysis in adult Japanese beetles is temporary in the laboratory but often fatal in the field [16,17]. Recovery from geranium/ QA-induced paralysis by Japanese beetles varied with time. A 1.0 μg dose of QA in an agar plug with 0.1 M sucrose was sufficient to paralyze 86% of the tested beetles (at 12 h), which was similar to what we observed for beetles feeding on geranium petals (87% at 12 h). However, at 24 h feeding duration, there was a significant reduction in the numbers of beetles paralyzed by geranium petals (59%) and QA (50%), i.e. beetle recovery. It is most likely that the enzymatic response at 24 h confers some resistance to geranium/QA intoxication, which is also reflected in the increased consumption rate of geranium petals or QA agar plugs by the beetles at 24 h. Potter and Held [17] also noted that Japanese beetles were able to consume a greater petal mass of zonal geranium upon recovery from geranium-induced torpidity. Between these periods (12 h and 24 h), geranium feeding resulted in upregulation of P450 and CoE activity while QA-feeding resulting in an increase in P450 and GST activity and a decline in CoE activity. P450, GST and CoE enzymes are likely involved in the recovery of the beetles from QA
Fig. 2. The effect of feeding duration on the paralysis of P. japonica by geranium petal, χ2 = 10.78 P = 0.0048.
There was a significant effect of feeding duration on the number of beetles that were paralyzed by geranium petals (χ2 = 10.78 P = 0.0048). The highest incidence of paralysis (87%) was observed in beetles that fed on geranium for 12 h. The number of beetles paralyzed at 6 h (70%) and 24 h (59%) were similar (P = 0.27) despite the rate of consumption of geranium petals increasing significantly between 12 h and 24 h. More than 50% of beetles that consumed geranium petals were paralyzed at each time point (6, 12 or 24 h) (Fig. 2). Consumption of geranium and rose petals induced activity of one or more enzyme groups (P450s, GSTs and CoEs) relative to non-feeding, but the magnitude of the enzyme induction varied by time and plant species i.e. significant interaction between diet and time for: P450 (F(4, 63) = 11.6, P < 0.0001), GST (F(4, 63) = 8.3, P < 0.0001) and CoE (F(4, 63) = 15.7, P < 0.0001). Feeding-induced enzyme activity was evident in beetles that consumed rose (P450, GST and CoE) or geranium petals (P450 and CoE) relative to the non-feeding control beetles. Generally, consumption of rose petals elicited different enzyme responses compared to consumption of zonal geranium petals. Rose induced higher enzyme activity relative to geranium at 6 and 12 h of feeding except for GST (Fig. 3A, B and C). Starvation did not induce P450 activity across time, while geranium induced significant P450 activity (2.3, 2 and 2.2-fold increases at 6, 12 and 24 h respectively) compared to starved beetles. Rose induced significant P450 activity (3 and 2-fold) at 6 h and 12 h but not at 24 h (Fig. 3A, F(2, 63) = 43, P < 0.0001). GST activity in starved beetles remained stable between 6 h and 12 h of feeding but significantly increased at 24 h. Interestingly, geranium did not induce significant GST activity at any measured time 4
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Fig. 3. A: P450 activity in P. japonica that consumed rose and geranium petals at different time periods. Diet (F(2, 63) = 43, P < 0.0001), Time (F(2, 63) = 6.5, P = 0.0026), Diet × Time (F(4, 63) = 11.6, P < 0.0001). B: GST activity in P. japonica that consumed rose and geranium petals at different time periods. Diet (F(2, P < 0.0001) Time (F(2, 45) = 1.6, P = 0.27) 45) = 42.7, Diet × Time (F(4, 45) = 8.3, P < 0.0001). C: CoE activity in P. japonica that consumed rose and geranium petals at different time periods. Diet (F(2, 63) = 3.4, P = 0.037), Time (F(2, 63) = 11.8, P < 0.0001), Diet × Time (F(5, 63) = 15.7, P < 0.0001). The bars on each treatment represent standard error of each mean. Letters on x axis denote treatment received by beetles: S, G and R represent the diet treatment of either no food, geranium or rose respectively. The corresponding numbers with each represent the duration of the exposure to the diet treatment (6, 12, or 24 h).
Fig. 5. The effect of feeding duration on the paralysis of P. japonica by quisqualic acid, χ2 = 13.4 P = 0.0019.
Fig. 4. Average mass (mg) of agar plugs with/without QA consumed by P. japonica at different time periods. Diet (F(1, 8) = 187, P < 0.0001), Time (F(1, 8) = 644, P < 0.0001), Diet × Time (F(1, 8) = 11.6, P < 0.001). The bars on each treatment represent standard error of each mean. Letters on x axis denote treatment received by beetles: A12-agar for 12 h, Q12-agar + QA for 12 h, A24-agar for 24 h, Q24-agar + QA for 24 h.
The optimal dose of QA that induced paralysis of Japanese beetle in this study (1.0 μg) is ten-fold higher compared to that of a previous study (0.1 μg) [18]. However, differences in experimental condition and design could be responsible for this variation. Aside from any cofounding factor, this suggests that different populations of Japanese beetles might vary in tolerance/susceptibility to QA. This form of phenotypic plasticity is typical of most naturally occurring arthropod populations to natural and synthetic pesticides [12,37]. Japanese beetle is an invasive pest in North America that gained entry in via the port of New Jersey in early nineteenth century [14]. Hence, it is likely that a century of geographic range expansion could lead to genetic variation resulting in phenotypic plasticity toward a naturally occurring but unique toxin like QA. The observed variation in enzyme (P450, GST and CoE) induction patterns by geranium and QA reflects differences in enzymes responses between the synthetic form of QA and natural forms in planta. Other phytochemicals present in planta (e.g. flavonoids, geraniol) may compete as substrates for detoxification enzymes in the midgut of P.
intoxication. This is because the activities of these enzymes generally peaked at 24 h after feeding on QA, while the incidence of paralysis significantly reduced. Previous studies [12,34,35] have also implicated detoxification enzymes in reducing the effects of toxic allelochemicals on insect herbivores. Transcriptomic studies [36] in other herbivoreplant systems have revealed feeding on novel or toxic host plants usually triggers a suite of detoxification enzymes, but very few or a single isoenzyme is actually responsible for circumventing the host plant toxin. Future in vitro studies will be required to understand the direct interaction of QA and the gut enzymes of Japanese beetles, and will provide further insight into which of these enzymes play vital roles in the metabolism of QA. Furthermore, analysis of the transcriptome of Japanese beetles will help identify the isoforms of detoxification enzymes that are involved in the response to geranium/QA intoxication.
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Fig. 6. A: P450 activity in P. japonica that consumed agar plugs with/without QA at different feeding duration. Diet (F(1,20) = 31, P < 0.09), Time (F(1, 20) = 13.5, P = 0.0015), Diet × Time (F(1, 20) = 1.18, P < 0.29). B: GST activity in P. japonica that consumed agar plugs with/without QA at different time periods. Diet (F(1, P = 0.045) Time (F(1, 20) = 18.3, P = 0.0004), 20) = 4.6, Diet × Time (F(1, 20) = 7.9, P = 0.01). C: CoE activity in P. japonica that consumed agar plugs with/without quisqualic at different time periods. Diet (F(1, 20) = 238, P < 0.0001) Time (F(1, 20) = 41, P = 0.0026), Diet × Time (F(1, 20) = 17.5, P < 0.0001). Letters on x axis denote treatment received by beetles: A12-agar for 12 h, Q12agar + QA for 12 h, A24-agar for 24 h, Q24-agar + QA for 24 h.
However, enzyme induction does not completely prevent toxicity of QA/zonal geranium indicating a mismatch between Pelargonium × hortorum chemistry and P. japonica's detoxification kit. Evolutionarily, it is not surprising that toxins in Pelargonium are novel to P. japonica since they share no natural history. Pelargonium spp. originate from South Africa [39] and the beetle, originating from Asia, would have experience with members of Geraniaceae but not Pelargonium spp. Future studies in this plant-insect interaction would benefit from comparisons between non-toxic plants in the Geraniaceae such as P. × scarboroviae [16] and the toxic hybrids of P. × hortorum.
japonica or inhibit detoxification protein induction. Hence, allelochemicals other than QA present in geranium petals possibly have an effect on detoxification enzymes or stress responses. Neal and Berenbaum [35] reported that gut enzymes from Papilio polyxenes were sensitive to phytosynergists like myristicin and safrole, present in umbellifers, which increases the toxicity of in planta furanocoumarins relative to synthetic forms. Francis et al. [38] also observed significant variation in the GST activity in aphids that were fed on Brassica plants and aphids that fed on allelochemicals typical of Brassicaceae (i.e. glucosinolate) incorporated into artificial diet. Further investigation is required to identify other phytochemicals present in geranium that could be acting as synergists for the toxicity of QA to P. japonica. Our experiment with agar plugs provides insight into enzymatic responses to QA intoxication independent of other phytochemicals. This experimental design allows for indirect comparison of enzyme activity from consumption in planta versus artificial doses of QA. We found that CoE activity was induced coincident with significant paralysis when beetles consumed agar plugs with QA (i.e. at 12 h). CoE activity also increased over time when beetles consumed geranium petals and was greatest at 24 h when paralysis decreased. The increase in P450 and GST activities are coincident with a decrease in paralysis at 24 h in the agar plug experiment, suggesting that induction of P450, GTS and CoE are all involved in P. japonica's physiological response to QA intoxication. Snyder and Glendinning [22] reported a similar response in tobacco budworms feeding on toxic nicotine, where increases in P450 activity coincided with increases in nicotine consumption and reduction in intoxication.
Acknowledgements Young's Plant Farm, Auburn AL, generously donated the geraniums used in this study. This work was supported by the Alabama Agricultural Experiment Station through Hatch funding from the National Institute of Food and Agriculture, USDA. Thanks to Amy Worthington, Nate Hardy, Paul Nabity and Fang Zhu for reviewing the earlier drafts of this manuscript and Ting Li, Youhui Gong and Emily Wine for technical support. References [1] G.S. Fraenkel, The raison d'etre of secondary plant substances, Science 129 (1959) 1466–1470. [2] R.I. Krieger, P.P. Feeny, C.F. Wilkinson, Detoxication enzymes in the guts of caterpillars: an evolutionary answer to plant defenses? Science 172 (1971) 579–581. [3] L.M. Schoonhoven, J.J. Van Loon, M. Dicke, Insect-Plant Biology, Oxford University Press, 2005 (on Demand). [4] J.A. Fordyce, A.A. Agrawal, The role of plant trichomes and caterpillar group size on growth and defence of the pipevine swallowtail Battus philenor, J. Anim. Ecol. 70 (2001) 997–1005. [5] S. Malcolm, L. Brower, Evolutionary and ecological implications of cardenolide sequestration in the monarch butterfly, Cell. Mol. Life Sci. 45 (1989) 284–295.
5. Conclusion The present study shows that QA in its pure form or in planta elicits induction of detoxification enzymes in the gut of adult P. japonica. 6
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