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Pesticide Biochemistry and Physiology 107 (2013) 188–199

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Pesticide Biochemistry and Physiology

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Effects of triazophos on biochemical substances of transgenic Bt rice andits nontarget pest Nilaparvata lugens Stål under elevated CO2

0048-3575/$ - see front matter � 2013 Published by Elsevier Inc.http://dx.doi.org/10.1016/j.pestbp.2013.07.002

⇑ Corresponding author. Fax: +86 01064807123.E-mail address: [email protected] (F. Ge).

Lin-Quan Ge a,b, Jin-Cai Wu a, Yu-Cheng Sun b, Fang Ouyang b, Feng Ge b,⇑a School of Plant Protection, Yangzhou University, Yangzhou 220059, PR Chinab State Key Laboratory of Integrated Management of Pest and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

a r t i c l e i n f o a b s t r a c t

Article history:Received 23 May 2013Accepted 2 July 2013Available online 12 July 2013

Keywords:TriazophosNilaparvata lugensElevated CO2

Biochemical substancesControl efficacy

The brown planthopper (BPH), Nilaparvata lugens Stål, is a serious rice pest throughout Asia. Recent out-breaks of N. lugens populations were mainly associated with the overuse of pesticides and resistance toinsecticides. Warmer global temperatures that are associated with anthropogenic climate change arelikely to have marked ecological effects on terrestrial ecosystems. However, the effects of elevated CO2

concentrations on the biochemical, physiological and nutrient quality of transgenic Bt rice that has beentreated with pesticides and on the control efficacy of the pesticides are not understood. The present studyinvestigated changes in soluble sugar content, free amino acid levels, oxalic acid levels, flavonoids levels,and triazophos residues in transgenic Bt rice (TT51) and the control efficacy of triazophos for N. lugensfollowing triazophos foliar spray under conditions of elevated CO2 (eCO2). Our findings showed thatthe soluble sugar content of TT51 treated with triazophos under eCO2 was significantly higher than thatunder ambient CO2 (aCO2) and also higher than that of the non-transgenic parent (MH63) under aCO2.However, the results for free amino acid levels were the opposite of those for soluble sugar levels. Theoxalic acid and flavonoid contents of rice plants significantly decreased with increases in triazophos con-centration, CO2 concentration, and days after treatment (DAT). The oxalic acid and flavonoid contents ofTT51 treated with triazophos under eCO2 were significantly lower than those under aCO2 and also lowerthan those of MH63 under aCO2. The residue concentration of triazophos varied with CO2 concentration,rice variety, and DAT. The residues in TT51 treated with 80 ppm of trizaopos under eCO2 were signifi-cantly lower than those under aCO2 and those in MH63 under aCO2. The survival rate of nymphs N. lugensin TT51 under eCO2 was significantly higher than that under aCO2 and that in MH63 under aCO2 at 1 DATor 15 DAT after the release of 2nd instars nymphs. These findings indicated that (1) for TT51, triazophosreduced the resistance of rice plants to N. lugens with an elevated CO2 concentration, as N. lugens con-sumed more phloem sap on TT51 plants; (2) triazophos dissipation in TT51 under eCO2 was significantlyfaster than that under aCO2 and that in MH63 under aCO2; (3) the control efficacy of triazophos for N.lugens significantly decreased under eCO2. The present findings provide important information for inte-grated pest management among transgenic varieties.

� 2013 Published by Elsevier Inc.

1. Introduction

Triazophos is a broad-spectrum organophosphate insecticideand acaricide with nematicidal properties [1,2]. It is used to con-trol aphids, bollworms, red spiders, fruit borers, leaf hoppers andcutworms on a variety of crops and is particularly effective inthe control of plant nematodes [3–8]. The brown planthopper(BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a ma-jor insect pest of rice in Asia. Chemical control of rice insectpests is still the first choice for pest management for many farm-ers. The recent outbreaks of N. lugens populations that occurred

in China and other Asian countries were associated with pesti-cide overuse and resistance to certain insecticides [9–12]. Pesti-cides may disrupt populations of natural enemies and affect thebalance of natural enemies and their hosts. Some insecticides atsub-lethal dosages stimulate the growth and productivity of N.lugens [10,13–17]. Furthermore, pesticides may affect insectsindirectly, by altering the host plants’ nutrition (e.g., free aminoacids and sucrose), growth, development, and physiological andbiochemical processes, and they can even lead to a resurgenceof the target pests [18–21], although some insecticides havebeen found not to influence the photosynthesis rates of alfalfa[22].

Atmospheric carbon dioxide (CO2) levels have risen steadilysince the start of the industrial revolution, from 280 to 387 ppm

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L.-Q. Ge et al. / Pesticide Biochemistry and Physiology 107 (2013) 188–199 189

today [23]. Current levels of atmospheric CO2 are expected to dou-ble within the next 100 years [24]. Numbers studies have exam-ined how various ecosystems might respond to changingatmospheric levels of this gas [25–27]. Profound impacts of ele-vated CO2 on terrestrial ecosystems [28,29], especially on thechemical composition and nutrient quality of plants, are expected[30,31]. Some investigations have shown that exposure to elevatedCO2 levels increases plant photosynthesis, growth, above-groundbiomass, leaf area, yield and carbon:nitrogen (C:N) ratios, particu-larly in C3 plants [32–35]. However, the effects of elevated CO2

concentrations on the biochemical, physiological and nutrientquality of transgenic Bt rice that has been treated with pesticidesand on the control efficacy of the pesticides are not understood.

The objectives of this study were to evaluate (1) the residuedynamics of the insecticide triazophos in different rice varietiesunder elevated CO2; (2) the change in chemical composition andnutrient quality of the transgenic Bt rice treated with triazophosunder elevated CO2; (3) the control efficacy of triazophos for thebrown planthopper N. lugens in different rice varieties treated withtriazophos under elevated CO2.

2. Materials and methods

2.1. Open-top chambers

The present experiment was performed using eight octagonal,open-top chambers (OTCs), each measuring 4.2 m in diameter, lo-cated at the Observation Station of the Global Change BiologyGroup, Institute of Zoology, Chinese Academy of Science (CAS) inXiaotangshan County, Beijing, China (40�110 N, 116�240 E). Theatmospheric CO2 concentration treatments (1) current atmo-spheric CO2 levels (375 ll/l) (‘‘ambient CO2’’, ‘‘aCO2’’) and (2) dou-bled ambient CO2 (750 ll/l) (‘‘elevated CO2’’, ‘‘eCO2’’). Four OTCswere used for each concentration treatment. CO2 concentrationswere monitored from the rice seedling stage to the end of theexperiment and were adjusted with an infrared CO2 analyzer (Ven-tostat 8102; Telaire, Goleta, CA, USA) once every 20 min to main-tain the CO2 concentrations. The automatic-control system foradjusting the CO2 concentration and the specifications for theOTC are detailed in Chen et al. [36].

2.2. Rice varieties and culture

The transgenic Bt rice line (TT51) and the non-transgenicparental indica rice line Minghui 63 (MH63) were selected forthe OTC experiment evaluation. TT51 is a transgenic rice lineexpressing a Bt fusion gene derived from cry1Ab and cry1Ac un-der the control of the rice actinI promoter [37]. The transgenic Btrice line was provided by Lin YongJun, National Key Laboratoryof Crop Genetic Improvement, Wuhan, China. Seeds were sownoutdoors in a standard rice-growing soil in cement tanks (height60 cm, width 100 cm and length 200 cm). When the seedlingsreached the six-leaf stage, they were transplanted into 16-cmdiameter plastic pots with four hills per pot and three plantsper hill and placed in OTCs. Each OTC contained 24 plastic pots(i.e., twelve plastic pots for MH63 and twelve plastic pots forTT51). The pots were randomly placed in each OTC and re-ran-domized daily to minimize position effects. From the six-leafstage to the end of experiment, pure CO2 mixed with ambientair was supplied to each of the OTCs in the elevated CO2 treat-ment group; in contrast, ambient air was supplied to the OTCsof the ambient CO2 treatment group. Rice plants at the tilleringstage were used in these experiments.

2.3. Insects culture and insecticide

The N. lugens used in the experiments were obtained form a lab-oratory population maintained in a greenhouse at an ecologicallaboratory at Yangzhou University, that was originally obtainedfrom the China National Rice Research Institute (CNRRI; Hangzhou,China). Before the experiments started, the N. lugens colony was al-lowed to reproduce for two generations in cement tanks under nat-ural condition in Beijing. Technical triazophos (87% [AI]) waspurchased from the Shenli Pesticide Co., Ltd., Ningguo, Anhui,China.

2.4. Treatment setup of triazophos spraying

Triazophos was dissolved in acetone. Ten percent of an emulsi-fier was added and diluted to three concentrations (20, 40, and80 ppm) with tap water based on previous results from a sublethaltest [38]. Rice plants at the tillering stage were sprayed using a Jac-to sprayer (Maquinas Agricolas Jacto S.A., Brazil) equipped with acone nozzle (1-mm diameter orifice, pressure 45 psi, flow rate300 ml/min), applying 20, 40, and 80 ppm of triazophos in100 ml of spray per pot. Control plants at the same stage weresprayed with the same amount of acetone and emulsifier. Eachtreatment and control was replicated four times. The treated andcontrol rice plant stems were collected at 7, 14, 21, and 28 daysafter treatment (7, 14, 21 and 28 DAT). The soluble sugar content,free amino acid levels, oxalic acid levels, and flavonoid levels in therice stem were measured. Triazophos residue was analyzed usingthe same sample at the same DAT. Rice plants at the tillering stagewere sprayed with 80 ppm of triazophos, and after 1 and 15 days,120 2nd instars nymphs were released per pot, respectively. Thenumber of surviving nymphs was recorded at 3, 7, and 10 daysafter release (3, 7, and 10 DAR). Each treatment and control wasreplicated four times. The treated and control plants were coveredwith cages (screen size: 80-mesh) to prevent insect movementthroughout this experiment.

2.5. Soluble sugar analysis

Soluble sugars were determined based on the anthrone reagentmethod [39]. To begin, 0.5 mg of fresh rice stem was cut into piecesand transferred to a large test tube. Then, 10 ml of distilled waterwas added, heated in a boiling water bath for 20 min, and cooleddown to room temperature. The supernatant was absorbed. Theextraction process was replicated three times. The extraction solu-tion was transferred to a 100 ml volumetric flask and mixed withdistilled water to achieve a total volume of 100 ml. Five millilitersof anthrone reagent per 1 ml of extraction solution was added. Themixture was heated in a boiling water bath for 10 min and thencooled down with tap water. The absorbency at a wavelength of620 nm was measured using a 722 spectrometer (The 3rd Anlalyt-ical Instrument Company of Shanghai, Shanghai, China). A standardcurve was drawn with sucrose.

2.6. Free amino acid analysis

Free amino acids were determined based on the method of nin-hydrin [40]. First, 0.2 g of fresh rice stem was homogenized with5 ml of 10% acetic acid using a mortar and pestle. The solutionwas filtered, and the filtered solution was transferred to a 100 mlvolumetric flask and mixed with distilled water to achieve a totalvolume of 100 ml. One milliliter of extraction solution was trans-ferred to a 25 ml volumetric flask, and 3.5 ml of ninhydrin bufferdeveloping solution was added. Then, 0.1 ml of 0.1% ascorbic acidsolution was added, and the mixture was thoroughly shaken. Themixture was heated in a boiling water bath for 20 min and then

190 L.-Q. Ge et al. / Pesticide Biochemistry and Physiology 107 (2013) 188–199

removed and cooled rapidly. Following this process, 10 ml of 80%ethanol was added, and water was then added for a total volumeof 25 ml. The absorbency at a wavelength of 570 nm was measuredusing a 722 spectrometer (The 3rd Anlalytical Instrument Com-pany of Shanghai, Shanghai, China). A standard curve was drawnwith glutamic acid.

2.7. Determination of oxalic acid

The trichloride titanium developing method was used to testthe oxalic acid content [41]. Fresh rice stems were cut, weighed,pulverized, and then filtered. Active carbon was added to thesupernatant for decolorizing, and the carbon was then separatedfrom the solution using centrifugation. Decolorizing was repeatedusing the above method until the solution reached a colorless ormilk-white state. Then, 0.15 ml of 1% trichloride titanium wasadded to 3 ml of the decolorized solution and centrifuged fordeveloping. Absorbance was measured at 400 nm using a 722 spec-trometer (The 3rd Analytical Instrument Company of Shanghai,Shanghai, China). A standard curve was constructed using 99.5%oxalic acid (Shanghai No. 4 Reagent Co. Ltd, Shanghai, China).

2.8. Determination of flavonoids

The flavonoid level was measured using the method of Zhuanget al. [42]. The fresh rice stem was cut into small pieces beforeextraction. Then, the fresh rice stem was dried in an electric ovenat 80 �C and then ground. One gram of rice stem materials wasweighed and placed into a 50 ml centrifuge tube. Next, 10 ml of60% ethanol was added to the tube, vibrated using a supersonic cellpulverizer (Ningbo Xin Yi Science Instrument Ltd. Co., Nibo, Zhe-jiang) for 30 min at 70 �C, and then centrifuged at 3000 rpm for10 min. The extraction process was replicated twice. The superna-tant was absorbed. The extraction solution was transferred to a25 ml volumetric flask, and 60% ethanol was added for a total vol-ume of 25 ml. To a new 25 ml volumetric flask, 1 ml of 5% sodiumnitrite and 1 ml of rice stem extraction solution were added andthoroughly shaken. After 5 min, 1 ml of 10% aluminum chloridewas added. After 6 min, 10 ml of 4% sodium hydroxide was added,30% ethanol was added for a total volume of 25 ml, and the mixturewas then shaken thoroughly. After 15 min, the absorbance wasmeasured at 400 nm using a 722 spectrometer (The 3rd AnalyticalInstrument Company of Shanghai, Shanghai, China). A standardcurve was constructed using rutin (Shanghai No. 4 Reagent Co.Ltd, Shanghai, China).

2.9. Quantification of triazophos residues in the different rice varietiesfor GC analysis

The residual level of the insecticide triazophos was determinedusing gas chromatography (GC) [43]. Twenty grams of fresh stemsfrom each rice variety and each treatment was used for the GCanalysis. Each sample was ground into a coarse meal with a vege-tation disintegrator. The extraction process followed the methoddescribed by Gong et al. [44], with slight modifications. Twentygrams of prepared rice stem was placed in a beaker, and 80 ml ofacetone was added. The samples were then left overnight, followedby extraction with surging for 1 h. The samples were filtered usingan 80-mm glass filter and air pump. The stem residue was filteredwith 100 ml of acetone. The filtrates were combined and trans-ferred to a funnel with 5 ml acetone, after which 200 ml of 5% so-dium chloride and 50 ml of dichloromethane were added andsurged for 2 min. The dichloromethane phase was collected afterpartitioning and transferred to a funnel with anhydrous sodiumsulfate. The resultant solution was then filtered with 10 ml ofdichloromethane. The water phase was extracted by surging twice

with dichloromethane (30 ml, 30 ml). All extracts were combinedand concentrated to near dryness on a rotary vacuum flash evapo-rator (Eyela Model NEIS). A small piece of absorbent cotton wasplaced in the bottom of the glass cleanup column (1 cm i.d.), anda 4-cm-thick layer of anhydrous sodium sulfate, 5 g of florisil, a1-g mixture of florisil and activated carbon (4:0.2), and another4-cm-thick layer of anhydrous sodium sulfate were successivelyadded. The ratio of the adsorbent sample weight was 30:100. Thecolumn was prewashed in 30 ml of APEM, and the first 15 mlresulting from elution was discarded. The concentrated samplesof rice stem were dissolved in 10 ml APEM, placed in the cleanup, and evaporated on a rotary evaporator at 30 �C until only 1–2 ml remained. The concentrated samples were transferred to aflask, and APEM was added to achieve a volume of 5 ml for theGC analysis. The GC analysis used a combination of publishedmethods [5,8]. The triazophos residues were estimated using aGC machine (Model HP-5890 II) equipped with a SGE BPX50 col-umn (50% phenyl equivalent polysilphenylene-siloxane;30 m � 0.53 mm i.d � 0.5-lm film thickness). Other GC parame-ters were as follows: oven temperature 280 �C, injection port tem-perature 300 �C, detector (NPD) temperature 300 �C, nitrogen gasflow 30 ml/min, hydrogen gas flow 3 ml/min, and air flow 79 ml/min. The relative retention time (Rt) for triazophos was observedat 6.8 min. We placed 10 mg of purified (87%) triazophos (ShenliPesticide Co., Ltd., Ningguo, Anhui, China) into a 100 ml flask andfilled it with acetone to a certain volume as a mother solution toestablish a standard curve. The solution was serially diluted withacetone into 0.1, 0.5, 1, 5, and 10 lg/ml standard solutions. Thepeak area of the 10 ll standard solution was measured. Therewas a positive linear relationship (Y = 366.89 + 40.112 X,R2 = 0.996) between the peak area (Y) for triazophos and its con-centration (X). The limit of detection (LOD) for triazophos was0.075 ng, and the limit of quantification (LOQ) was 0.05 mg/kg.

2.10. Statistical analysis

Data were evaluated for normality and homogeneity of varianceusing Bartlett’s test [45]. Based on these evaluations, no transfor-mations were needed. Soluble sugar content, free amino acid lev-els, oxalic acid levels, flavonoid levels, insecticide triazophosresidues in the different rice varieties and the number of survivingN. lugens nymphs were analyzed using an analysis of variance (AN-OVA) with three factors (CO2 concentration, rice variety, insecti-cide concentration). Multiple comparisons of the means wereconducted using Fisher’s Protected Least Significant Difference(PLSD) test. All analyses were conducted using the GLM procedure(SPSS Inc., 2002) [46].

3. Results

3.1. Changes in triazophos-induced soluble sugar content and freeamino acid content in the different rice varieties under elevated CO2

The three-way ANOVA of the data, as shown in Fig. 1, showedthat insecticide concentration, rice variety and CO2 concentrationsignificantly influenced the soluble sugar content of the rice plants(Table 1). The soluble sugar content of the rice plants significantlyincreased with an increase in the triazophos concentration for bothMH63 and TT51 at different DATs, whether it was measured underaCO2 or eCO2. However, no significant difference between the 40and the 80 ppm triazophos treatments at 7, 14, 21, and 28 DATwas found. For two concentrations of CO2, grand means (meansof main effect) of the soluble sugar content of the rice plants trea-ted with triazophos under eCO2 was significantly higher than thatof rice plants under aCO2, increasing by 28.9%, 30.5%, 29.6%, and

Fig. 1. Changes in the soluble sugar content of rice treated with triazophos at the tillering stage for Minghui 63 (MH63) and TT51 (Transgenic) in ambient CO2 (aCO2) andelevated CO2 (eCO2) environments. Bars with different letters (two groups were compared between MH63 and TT51 under aCO2 or under eCO2, respectively) within thegraphs are significantly different at the 5% level. Each treatment and control was replicated four times.


25.5% at 7, 14, 21, and 28 DAT, respectively. For two rice varieties,grand means of the soluble sugar content of TT51 treated with tria-zophos was significantly higher than that of MH63, increasing by37.4%, 43.6%, 39.7%, and 23.0% at 7, 14, 21, and 28 DAT, respec-tively. Multiple comparisons of the means indicated that the solu-ble sugar content was significantly higher in the rice plants treatedwith all concentrations of triazophos than in the untreated con-trols; the soluble sugar content was significantly higher in riceplants under eCO2 than in those under aCO2.

The three-way ANOVA of the data, as shown in Fig. 2, showedthat insecticide concentration, rice variety and CO2 concentrationsignificantly influenced the free amino acid content of rice plantsunder aCO2 or eCO2 (Table 1). Similarly, the free amino acid con-tent of the rice plants significantly increased with an increase inthe triazophos concentration for both MH63 and TT51 at differentDATs, whether it was measured under ambient CO2 or elevatedCO2. However, no significant difference in the free amino acid con-tent between the 40 ppm and the 80 ppm triazophos treatmentswas found. For two concentrations of CO2, grand means of the freeamino acid content of rice plants treated with triazophos undereCO2 was significantly lower than that of rice plants under aCO2,decreasing by 21.6%, 23.4%, 28.9%, and 13.9% at 7, 14, 21, and 28DAT, respectively. For two rice varieties, grand means of the freeamino acid content in TT51 treated with triazophos was signifi-cantly lower than that of MH63, decreasing by 27.9%, 32.0%,28.3%, and 25.7% at 7, 14, 21, and 28 DAT, respectively. Multiplecomparisons of the means indicated that the free amino acid con-tent was significantly higher in the rice plants treated with all

concentrations of triazophos than in the untreated controls; thefree amino acid content was significantly lower in rice plants undereCO2 than in those under aCO2.

3.2. Effect of triazophos on oxalic acid and flavonoid levels for differentrice varieties under elevated CO2

The three-way ANOVA of the data, as shown in Fig. 3, showedthat insecticide concentration, rice variety and CO2 concentrationsignificantly influenced the oxalic acid content of rice plants underaCO2 or eCO2 (Table 2). The oxalic acid content of rice plants signif-icantly decreased with an increase in the triazophos concentrationfor both MH63 and TT51 at different DATs for both ambient CO2

and elevated CO2. However, no significant difference between the40 and the 80 ppm triazophos treatments at 7, 14, 21, and 28DAT was found. For two concentrations of CO2, grand means ofthe oxalic acid content of the rice plants treated with triazophosunder eCO2 was significantly lower than that of rice plants underaCO2, decreasing by 29.8%, 26.2%, 30.9%, and 36.2% at 7, 14, 21,and 28 DAT, respectively. For two rice varieties, grand means ofthe oxalic acid content in TT51 treated with triazophos was signif-icantly lower than that of MH63, decreasing by 13.6%, 9.3%, 10.5%,and 8.7% at 7, 14, 21, and 28 DAT, respectively. Multiple compari-sons of the means indicated that the oxalic acid content was signif-icantly lower in the rice plants treated with all concentrations oftriazophos than in the untreated controls; the oxalic acid contentwas significantly lower in rice plants under eCO2 than in those un-der aCO2.

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Fig. 2. Effects on the amino acid content of insecticide triazophos treated rice at the tillering stage for Minghui 63 (MH63, no-transgenic) and TT51 (Transgenic) in ambientCO2 (aCO2) and elevated CO2 (eCO2) environments. Bars with different letters (two groups were compared between MH63 and TT51 under aCO2 or eCO2, respectively) withinthe graphs are significantly different at the 5% level. Each treatment and control was replicated four times.

Table 1Analysis of variance of soluble sugar and amino acid data shown in Figs. 1 and 2.

DAT Source of variance Df Soluble sugar Amino acid

F-value P-value F-value P-value

7 CO2 concentration (A) 1 365.8 0.0001 230.0 0.0001Rice variety (B) 1 568.3 0.0001 414.3 0.0001Insecticide concentration (C) 3 411.9 0.0001 974.5 0.0001A � B 1 59.0 0.0113 35.9 0.0051A � C 3 29.0 0.0001 20.1 0.0001B � C 3 23.4 0.0001 30.8 0.0525A � B � C 3 5.2 0.0036 3.0 0.0378

14 CO2 concentration (A) 1 576.4 0.0001 689.1 0.0001Rice varieties (B) 1 1054.5 0.0001 1431.8 0.0001Insecticide concentration (C) 3 733.5 0.0001 2176.5 0.0001A � B 1 121.0 0.0001 133.2 0.0001A � C 3 27.2 0.0001 39.2 0.0001B � C 3 28.4 0.0001 82.3 0.0001A � B � C 3 8.5 0.0001 15.9 0.0001



DAT is days after treatment. Df is degrees of freedom.


Table 2Analysis of variance of oxalic acid and flavonoid content data shown in Figs. 3 and 4.

DAT Source of variance Df Oxalic acid Flavonoids







Fig. 3. Effects on the oxalic acid content of triazophos treated rice at the tillering stage for Minghui 63 (MH63) and TT51 (Transgenic) in ambient CO2 (aCO2) and elevated CO2

(eCO2) environments. Bars with different letters (two groups were compared between MH63 and TT51 under aCO2 or eCO2, respectively) within the graphs are significantlydifferent at the 5% level. Each treatment and control replicated was four times.


Fig. 4. Effects on the flavonoid content of triazophos treated rice at the tillering stage for Minghui 63 (MH63) and TT51 (Transgenic) in ambient CO2 (aCO2) and elevated CO2

(eCO2) environments. Bars with different letters (two groups were compared between MH63 and TT51 under aCO2 or eCO2, respectively) within the graphs are significantlydifferent at the 5% level. Each treatment and control was replicated four times.


The three-way ANOVA of the data, as shown in Fig. 4, showedthat insecticide concentration, rice variety and CO2 concentrationsignificantly influenced the flavonoid content of the rice plants un-der aCO2 or eCO2 (Table 2). Similarly, the flavonoid content of therice plants significantly decreased with an increase in the triazo-phos concentration for both MH63 and TT51 at different DATs forboth ambient CO2 and elevated CO2. However, no significant differ-ence between the 40 and the 80 ppm triazophos treatments at 7,14, 21, and 28 DAT was found. For two concentrations of CO2,grand means of the flavonoid content of the rice plants treatedwith triazophos under eCO2 was significantly lower than that ofrice plants under aCO2, increasing by 17.7%, 21.7%, 26.1%, and18.1% at 7, 14, 21, and 28 DAT, respectively. For two rice varieties,grand means of the flavonoid content of TT51 treated with triazo-phos was significantly lower than that of MH63, increasing by 9.7%,11.5%, 11.3%, and 4.6% at 7, 14, 21, and 28 DAT, respectively. Multi-ple comparisons of the means indicated that flavonoid content wassignificantly lower in the rice plants treated with all concentrationsof triazophos than in the untreated controls; the flavonoid contentwas significantly lower in rice plants under eCO2 than in those un-der aCO2.

3.3. Elevated CO2 affects triazophos residue in the different ricevarieties

The three-way ANOVA of the data, as shown in Fig. 5, showedthat there were significant differences between MH63 and TT51

under aCO2 and eCO2 (Table 3). The residue content of the insecti-cide triazophos in the rice plants significantly decreased with theincrease in the DAT for both MH63 and TT51 and under bothaCO2 and eCO2 (Fig. 5). However, the residue content under eCO2

was not measured in MH63 and TT51 treated with 20 and40 ppm of triazophos at 28 DAT. For two concentrations, grandmeans of the residue of triazophos in the rice plants under eCO2

was significantly lower than that of rice plants under aCO2,decreasing by 28.6%, 35.4%, 45.3%, and 67.3% at 7, 14, 21, and 28DAT, respectively. For two rice varieties, grand means of the resi-due of triazophos was significantly lower in TT51 than in MH63,decreasing by 24.0%, 34.1%, 36.6%, and 39.0% at 7, 14, 21, and 28DAT, respectively.

3.4. Number of surviving N. lugens nymphs treated with triazophos inthe different rice varieties under elevated CO2

The three-way ANOVA of the data, as shown in Fig. 6, showedthat insecticide concentration, rice variety and CO2 concentrationsignificantly influenced the number of surviving N. lugens nymphs(Table 4). The number of surviving N. lugens nymphs significantlydecreased with an increase in the DAR for both MH63 and TT51after 1 DAT under both ambient CO2 and elevated CO2. For twoconcentrations of CO2, grand means of the number of survivingN. lugens nymphs on rice plants treated with 80 ppm of triazophosunder eCO2 was significantly higher than that of rice plants underaCO2, increasing by 9.1%, 12.4% and 14.8% at 3, 7 and 10 DAR,

Table 3Analysis of variance of insecticide triazophos residues data shown in Fig. 5.

DAT Source of variance Df F-value P-value

7 CO2 concentration (A) 1 511.4 0.0001Rice varieties (B) 1 341.4 0.0001Insecticide concentration (C) 3 1604.0 0.0001A � B 1 2.1 0.1575A � C 3 69.7 0.0001B � C 3 100.5 0.0001A � B � C 3 1.2 0.3234






respectively. For two rice varieties, grand means of the number ofsurviving N. lugens nymphs on TT51 treated with 80 ppm of triazo-phos was significantly higher than that in MH63, increasing by12.3%, 17.6%, and 20.1% at 3, 7, and 10 DAR, respectively. Multiplecomparisons of the means indicated that the number of survivingN. lugens nymphs on different rice varieties was significantly lowerfor those treated with 80 ppm of triazophos than it was for the un-treated controls; the value under eCO2 was significantly higherthan that under aCO2.

The three-way ANOVA of the data, as shown in Fig. 6, showedthat insecticide concentration, rice variety and CO2 concentrationsignificantly influenced the number of surviving N. lugens nymphs(Table 4). Similarly, the number of surviving N. lugens nymphs sig-nificantly decreased with an increase of DAR for both MH63 andTT51 after 15 DAT for both ambient CO2 and elevated CO2. Fortwo concentrations, grand means of the number of surviving N. lu-gens nymphs on rice plants treated with 80 ppm of triazophos un-der eCO2 was significantly higher than that of rice plants underaCO2, increasing by 5.3%, 8.8% and 9.7% at 3, 7 and 10 DAR, respec-tively. For two rice varieties, grand means of the number of surviv-ing N. lugens nymphs on TT51 treated with 80 ppm of triazophoswas significantly higher than that in MH63, increasing by 7.6%,10.6%, and 12.8% at 3, 7, and 10 DAR, respectively. Multiple com-parisons of the means indicated that the number of surviving N. lu-gens nymphs was significantly lower for the different rice varietiestreated with 80 ppm of triazophos than for the untreated controls;the number of surviving N. lugens nymphs was significantly higheron rice plants under eCO2 than on those under aCO2.

Fig. 5. Dynamics of triazophos residue after treatment in rice at the tillering stage for minghui 63 (MH63) and TT51 (Transgenic) in ambient CO2 (aCO2) and elevated CO2

(eCO2) environments. Bars with different letters (two groups were compared between MH63 and TT51 under aCO2 or eCO2, respectively) within the graphs are significantlydifferent at the 5% level. Each treatment and control was replicated four times.

Fig. 6. Comparison of the number of surviving nymphs N. lugens on rice plants after 1 (A) and 15 DAT (B) at the tillering stage for Minghui 63 (MH63) and TT51 (Transgenic) inambient CO2 (aCO2) and elevated CO2 (eCO2) environments. DAT is days after treatment. Bars with different letters (two groups were compared between MH63 and TT51under aCO2 or under eCO2, respectively) within the graphs are significantly different at the 5% level. Each treatment and control was replicated four times. In all, 120 nymphswere released per treatment. DAR is days after nymph release.


4. Discussion

Elevated CO2 can lead to a significant increase in plant photo-synthesis, growth, aboveground biomass, leaf area, yield and car-bon: nitrogen (C:N) ratios, particularly in C3 plants [34,47–49].All these changes in the chemical components of plants result fromelevated CO2 and, in turn, affect the production of secondarymetabolites [50–52]. Our findings indicated that the soluble sugarcontent of TT51 with triazophos treatment under eCO2 was signif-icantly higher that of MH63 under aCO2 (Fig. 1). Previous findingsindicated that pesticide treatment altered the physiological andbiochemical changes of rice plants and consequently affected thesurvival and feeding rates of N. luges and the damage level of plants

[18,19,53,54]. Gu et al. [55] and Liu et al. [56] reported that Chilosupperssalis (Walker) preferred feeding on rice plants because oftheir high sugar content. The current findings showed that triazo-phos treatment would facilitate the feeding of pests on phloem sapin TT51 under eCO2. The flight capacity of N. lugens adults thatwere developed from nymphs feeding on rice plants treated withinsecticides (including triazophos) was enhanced, indicating thattreated rice plants supply more energy for flight in N. lugens [57].Shen and Cheng [58] revealed that the fecundity of N. lugens adultfemales significantly increased after long-distance migration.

Free amino acids in rice plants could be directly used as a nitro-gen nutrition source for N. lugens [59]. Our results showed that thefree amino acid content of rice plants significantly increased with

Table 4Analysis of variance of the number of surviving effects of insecticide triazophos residues on nymphs N. lugens data shown in Fig. 6.

DAR Source of variance Df 1 DAT 15 DAT





DAR is days after nymph release. Df is degrees of freedom. DAT is days after treatment.


an increase in the triazophos concentration for both MH63 andTT51 at different DATs under both ambient CO2 and elevated CO2

(Fig. 2). It has been found that decamethrin decreases the ratio ofcarbohydrates to nitrogen and increases the level of free aminoacids in susceptible rice strains [60]. The free amino acid contentof rice plants treated with insecticides was significantly increased[61]. An increase in free amino acids and a decrease in the C/N ratioare important factors for stimulating feeding in N. lugens. The ami-no acid content in resistant varieties was lower than that in sus-ceptible varieties [62–64]. Sun et al. [65] reported that loweramounts of amino acids were found in cotton phloem undereCO2 than under aCO2 levels. Higher amounts of free amino acidswere found in Aphis gossypii fed cotton grown in eCO2 than thosefed cotton grown in aCO2. These findings indicate that Aphis gos-sypii will consume more phloem sap to meet their nutritionalrequirements in an aCO2 environment, which will balance aminoacid content in the plants [65].

Flavonoids are important secondary compounds that are asso-ciated with a higher plant resistance to insects. Flavonoids mayresult in the deterrence, antifeeding, and toxicosis of insects[66–68]. The present results indicated that the oxalic acid andflavonoid content of TT51 treated with triazophos under eCO2

was significantly lower than that of MH63 under aCO2 (Figs. 3and 4). Flavonoids from the wheat plant significantly suppressedthe growth, development, and reproduction of Sitobion avenae[69]. Grayer et al. [83] reported that the extracted isolated flavo-noids from the stems of anti-insect rice have an antifeeding ef-fect on N. lugens; therefore, high amounts of flavonoids werean important factor in determining rice resistance to N. lugens.Researchers have revealed that resistant varieties of rice containhigher amounts of three flavonoids than sensitive varieties[70,71]. When spread on a susceptible rice plant (TN1), tricin(500 lg/ml) significantly inhibited the oviposition and feedingbehavior of brown planthopper females in choice tests [72]. Oxa-lic acid is the most active anti-feedant administered as a freeacid or salt [73] and is considered to be an important factor inthe resistance to N. lugens in rice [74]. Wu et al. [41] reportedthat a decrease in the oxalic acid content of rice plants after fun-gicide jingganmycin and insecticide bisultap treatments resultedin a decreased rice resistance to N. lugens. Applications of several

pesticides, including insecticides, fungicides and herbicides, re-duce the resistance of rice to N. lugens [18,19]. The oxalic acidin rice plants after two selective insecticide (imidacloprid andbuprofezin) treatments was significantly decreased and resultedin Typoryza incertulas (Walker) populations of resurgence [75].Our results indicated that the treatment of TT51 with triazophossignificantly decreased its resistance to N. lugens under eCO2.

The residue of triazophos was significantly lower on thedifferent rice varieties under eCO2 than on those under aCO2.Triazophos residue in TT51 under eCO2 was significantly lowerthan that in MH63 under aCO2 (Fig. 5). The control efficacy oftriazophos for N. lugens was consistent with the dynamics oftriazophos residue at both 1 DAT and 15 DAT (Fig. 6). Thecurrent results indicated that the triazophos dissipation rate inTT51 under eCO2 was significantly faster than that in MH63under aCO2. With an elevated CO2 concentration, the control effi-cacy of triazophos decreases. On the whole, the resistance of thetransgenic Bt rice variety (TT51) treated with triazophos toN. lugens may decrease under eCO2. However, Chen et al.[76]reported that the fecundity of N. lugens on the Bt rice (Cry1Ab)under aCO2 was significantly decreased in every generation,compared to the non-Bt rice in the field investigation, and didnot result in an outbreak of its non-target herbivore (N. lugens).

The present results showed that TT51 treatment with triazo-phos reduced the resisance of rice plants to N. lugens under eCO2,and triazophos dissipation in TT51 under eCO2 significantly fasterthan that in MH63 under aCO2.Therefore, we suggest that recom-mendation rate of triazophos need to be increased when the insec-ticide is used for control of rice pests under eCO2. However, themechanisms leading to the effects of transgenic Bt rice or pesti-cide-treated Bt rice varieties under elevated CO2 on the fecundityof N. lugens need to be further investigated.

Acknowledgments

This research was supported in part by the State Key Laboratoryof Integrated Management of Pest Insects and Rodents (Grant No.Chinese IPM1202) and by the National Natural Science Fund of Chi-na (31201507 and 31221091) and Post-doctoral Fund of China(2012M520378).


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