Interactive Effects of Elevated CO2 and Temperature on Rice Planthopper, Nilaparvata lugens

Journal of Integrative Agriculture2014, 13(7): 1520-1529 July 2014RESEARCH ARTICLE

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.doi: 10.1016/S2095-3119(14)60804-2

Interactive Effects of Elevated CO2 and Temperature on Rice Planthopper, Nilaparvata lugens

SHI Bao-kun, HUANG Jian-li, HU Chao-xing and HOU Mao-lin

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

Abstract

It is predicted that the current atmospheric CO2 concentration will be doubled and global mean temperature will increase by 1.5-6°C by the end of this century. Although a number of studies have addressed the separate effects of CO2 and temperature on plant-insect interactions, few have concerned with their combined impacts. In the current study, a factorial experiment was carried out to examine the effect of a doubling CO2 concentration and a 3°C temperature increase on a complete generation of the brown planthopper (Nilaparvata lugens) on rice (Oryza sativa). Both elevated CO2 and temperature increased rice stem height and biomass of stem parts. Leaf chlorophyll content increased under elevated CO2, but only in ambient temperature treatment. Water content of stem parts was reduced under elevated temperature, but only when coupled with elevated CO2. Elevated CO2 alone increased biomass of root and elevated temperature alone enhanced leaf area and reduced ratio of root to stem parts. Brown planthopper (BPH) nymphal development was accelerated, and weight of and honeydew excretion by the F1 adults was reduced under elevated temperature only. Longevity of brachypterous females was affected by a significant interaction between CO2 and temperature. At elevated temperature, CO2 had no effect on female longevity, but at ambient temperature, the females lived shorter under elevated CO2. Female fecundity was higher at elevated than at ambient temperature and higher at elevated CO2 than at ambient CO2. These results indicate that the combined effects of elevated temperature and CO2 may enhance the brown planthopper population size.

Key words: climate change, elevated CO2, global warming, Nilaparvata lugens, rice, development

INTRODUCTION

Atmospheric CO2 concentrations are predicted to double the current levels by the end of this century. As a conse-quence, a 1.5-6°C increase in global mean temperature is also predicted (IPCC 2001). Such global-scale climatic changes will potentially alter plant-herbivore interactions through effects on plant and insect growth, development, and survival.

It is known that enhanced CO2 increases plant photo-

synthetic rate (Norby et al. 1999) and biomass (Owensby et al. 1999), which reduces foliar nitrogen concentrations and thereby increases C/N ratio in leaves (Pritchard et al. 1999; Hughes and Bazzaz 2001; Goverde and Erhardt 2003). The increased plant growth and increase in C/N ratio can reduce the nutritional value of plant tissues for insect herbivores and negatively influence herbivore per-formance (Goverde and Erhardt 2003; Barbehenn et al. 2004; Asshoff and Hättenschwiler 2005). Responses of herbivorous insects to elevated CO2 are species-specific (Hillstrom et al. 2010); the majority of chewing insect herbivores (such as Lepidoptera) is adversely affected

Received 6 September, 2013 Accepted 29 November, 2013Correspondence HOU Mao-lin, Tel/Fax: +86-10-62833985, E-mail: [email protected]

Interactive Effects of Elevated CO2 and Temperature on Rice Planthopper, Nilaparvata lugens 1521

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

(Goverde and Erhardt 2003; Barbehenn et al. 2004; Knepp et al. 2007), and compensatory consumption often occurs in these insects (Barbehenn et al. 2004). However, the responses of sucking insect herbivores to elevated CO2 are varying (Sun and Ge 2011; Robinson et al. 2012). For example, some aphid species perform better (Bezemer et al. 1998; Gao et al. 2008) while others show no change in performance or even show decreased performance (Docherty et al. 1997) under elevated CO2 conditions. Still, even the same aphid species shows different and varying results at population level as compared with individual level (Bezemer et al. 1998) or on different host plants (Awmack et al. 1997; Sudderth et al. 2005) or on different genotypes of the same plant species (Guo et al. 2013). Under elevated CO2 concentrations, herbivorous insect development and survival are mostly expected to decrease due to reduced host plant’s nutritional value (Johns and Hughes 2002).

Increased temperature also affects plants directly. As temperature increases, rates of vegetative development and maturation increase, resulting in short life cycle in annual plants (Rawson 1992). Insects are poikilo-therms and increased temperature directly accelerates development in most insects. Shortened development duration may increase numbers of generations and increase population size more rapidly for multivoltine species in the growing season. Insect species that are currently restricted in distribution owing to cold winter temperature may also extend their ranges into new areas as winter become milder (Parmesan et al. 1999). Increased temperatures are therefore expected to favour insect survival, particularly in cooler climates.

It is well recognized that temperature and atmo-spheric CO2 concentrations will increase concurrently, and will either directly or indirectly affect plant-insect interactions, but the effects of these two factors have been studied in combination in much fewer cases than that they have been examined separately (Johns and Hughes 2002; DeLucia et al. 2013). A critical question here is that the individual effects of CO2 and temperature may be altered when the two factors are combined, and focus on individual effects of CO2 and temperature cannot provide a real picture of the effects of future climate change on plant-insect interactions (DeLucia et al. 2013). Plant growth and phenology are known to be affected by the interaction between

CO2 and temperature (Rawson 1992; Morison and Lawlor 1999; Cheng et al. 2009; Zhang et al. 2013). Warm temperature may decrease the accumulation of carbohydrates in some species, and the stimulation of plant growth by elevated CO2 may decrease or cease altogether at very cool temperature (Morison and Lawlor 1999). By contrast, the interactive effects of CO2 and temperature on herbivorous insects have not received much attention (Robinson et al. 2012; DeLucia et al. 2013). Buse and Good (1996) revealed that elevated temperature accelerated development of eggs and larvae, but delayed emergence of adults in the winter moth Operophtera brumata whilst elevated CO2 had no effects on any of the parameters measured. In contrast, a study directed to the gypsy moth Lymantria dispar found that elevated CO2 reduced growth, but temperature had no effects (Williams et al. 2000). Neither of these studies measured the growth and de-velopment of the insects over a complete generation under the experimental conditions. Johns and Hughes (2002) reported that development time of a leaf-miner Dialectica scalariella was shortened for about 14 d in climatic change conditions as compared to the current ambient conditions, and that elevated CO2 had a strong negative effect on larval survival and adult weight when combined with an elevated temperature treatment. For the beetles Octotoma championi and O. scabripennis, Johns et al. (2003) showed that development time was accelerated by approximately 10-13 d at the higher temperature, but was not affected by CO2; adult weight and consumption rates of free-living beetles were not affected by either CO2 or temperature.

The brown planthopper Nilaparvata lugens (Stål) (Homoptera: Delphacidae) is a phloem-sucking insect pest on rice Oryza sativa L. (Graminales: Poaceae) throughout Asia (Duck and Thomas 1979). The brown planthopper is dimorphic, with fully winged ‘macropter-ous’ and truncate-winged ‘brachypterous’ forms (Iwan-aga et al. 1985). Both nymphs and adults aggregate and feed on leaf sheaths at the basal portion of rice plants (Sogawa and Cheng 1979). To our knowledge, there have been no previous studies of interactive effects of temperature and CO2 on this important crop pest (Hu et al. 2010; Wang et al. 2010).

In this study, effects of CO2 and temperature on per-formance of the brown planthopper N. lugens feeding

1522 SHI Bao-kun et al.


on rice plants were examined using a factorial design. One complete generation of the herbivore was grown on rice in each of four CO2×temperature combinations, which was aimed to determine the independent and in-teractive effects of elevated CO2 and temperature on the development and fecundity of the brown planthopper.

RESULTS

Rice plant development

Both elevated CO2 and temperature promoted stem elon-gation, and there was a significant interaction between the two factors on stem height of rice plants exposed to the four treatments for 50 d (Table). Rice stems grown under elevated CO2 and temperature were higher than those at the other three treatments, and those in ambient CO2 and temperature treatment were shorter than those in the other three treatments (Fig. 1-A).

There was a significant influence of temperature on leaf area; either CO2 concentration or its interaction with temperature had no significant effects (Table). Rice leaves in elevated temperature treatments were on aver-age 216 mm2 greater than those in ambient temperature treatments (Fig. 1-B).

Both fresh and dry weight of stem parts was signifi-cantly affected by CO2 concentration and temperature, but not by their interaction (Table). Fresh weight of stem parts increased significantly in elevated temperature treatments and in elevated CO2 treatments compared with their corresponding ambient treatments (both by about 53%) (Fig. 1-C), and dry weight was 76.5 and 56.5% more in elevated temperature treatments and in elevated CO2 treatments than their respective ambient treatments, respectively (Fig. 1-D).

There was a significant interaction between the effects of CO2 concentration and temperature on water content of stem parts (Table). In ambient CO2 treatments, there was no difference in water content of stem parts between the two temperature levels, while in elevated CO2 treatments, water content at elevated temperature was 5.3% lower than that at ambient temperature (Fig. 1-E).

CO2 concentration had significant influence on dry weight of root; either temperature or its interaction with CO2 concentration had no significant effects (Table).

Roots of plants in elevated CO2 treatments were 40.4% heavier than those in ambient CO2 treatments (Fig. 1-F).

Ratio of root to stem parts was significantly affected by temperature, but not by CO2 concentration and its interaction with temperature (Table). The ratio was 36.1% lower in elevated temperature treatments than in ambient temperature treatments (Fig. 1-G).

Leaf chlorophyll content was significantly influenced by the interaction between CO2 concentration and tem-perature (Table). In ambient temperature treatments, chlorophyll content was 24.7% higher at elevated CO2

than at ambient CO2, while in elevated temperature treatments, there was no difference in chlorophyll content between the two CO2 concentrations (Fig. 1-H).

Table Summary of analysis of variance (ANOVA) results for effects of CO2 and temperature on plant and insect performanceMeasurement Treatment F (df) PPlants

Stem height CO2 46.933 (1, 80) <0.001Temperature 31.383 (1, 80) <0.001

CO2×Temperature 6.351 (1, 80) 0.014Leaf area CO2 3.787 (1, 80) 0.055

Temperature 28.832 (1, 80) <0.001CO2×Temperature 0.158 (1, 80) 0.692

Fresh weight of stem parts CO2 31.331 (1, 80) <0.001Temperature 31.097 (1, 80) <0.001

CO2×Temperature 0.064 (1, 80) 0.801Dry weight of stem parts CO2 29.637 (1, 80) <0.001


Water content of stem parts CO2 0.324 (1, 80) 0.571Temperature 22.623 (1, 80) <0.001

CO2×Temperature 4.497 (1, 80) 0.037Dry weight of root CO2 23.591 (1, 80) <0.001

Temperature 2.006 (1, 80) 0.161CO2×Temperature 0.080 (1, 80) 0.777

Ratio of root to stem parts CO2 2.554 (1, 80) 0.114Temperature 52.858 (1, 80) <0.001

CO2×Temperature 0.008 (1, 80) 0.928Leaf chlorophyll content CO2 22.433 (1, 80) <0.001

Temperature 1.800 (1, 80) 0.184CO2×Temperature 16.234 (1, 80) <0.001

PlanthoppersNymphal duration CO2 0.460 (1, 229) 0.498


Adult weight CO2 2.068 (1, 166) 0.152Temperature 4.318 (1, 166) 0.039

CO2 ×Temperature 2.704 (1, 166) 0.102Female longevity CO2 3.749 (1, 73) 0.057


No. of eggs per female CO2 7.158 (1, 73) 0.009Temperature 9.160 (1, 73) 0.003

CO2×Temperature 0.241 (1, 73) 0.625Honeydew excretion CO2 2.229 (1, 60) 0.141




Development of F1 generation of the planthopper

When the planthoppers were exposed as newly hatched nymphs to rice plants grown in the four treatments, elevated temperature reduced nymphal duration, CO2 concentration and its interaction with temperature showed no significant influence (Table). Adults in elevated temperature treatments emerged on average 1.1 d earlier at ambient CO2 and 1.3 d earlier at elevated CO2 than those at the corresponding CO2 levels in am-bient temperature treatments (Fig. 2-A).

The adults emerged from the nymphs feeding on rice plants in elevated temperature treatments showed reduced weight (by 7.6%), compared to those in ambient temperature treatments (Table and Fig. 2-B).

Longevity and fecundity of F1 generation of the planthopper

Adults emerged from the nymphs feeding on rice plants grown in the four treatments were used to further test the effects of CO2 concentration and temperature on

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Fig. 1 Biological parameters of rice plants grown in four CO2×Temperature treatments. A, stem height. B, leaf area. C, fresh weight of stem parts. D, dry weight of stem parts. E, water content of stem parts. F, dry weight of root. G, ratio of root to stem parts. H, relative chlorophyll content. n=20 for each treatment. aCO2+aT, ambient CO2 and temperature; aCO2+eT, ambient CO2 and elevated temperature; eCO2+aT, elevated CO2 and ambient temperature; eCO2+eT, elevated CO2 and temperature. Different letters are labeled over the bars in a panel to denote significant difference between treatments (Tukey HSD test, P=0.05). The same as below.



longevity and fecundity of females at their originating treatments. There was a significant interactive effect of CO2 and temperature on female longevity (Table). In ambient temperature treatments, females lived 4.4 d shorter at elevated than at ambient CO2, while in elevated temperature treatments, there was no difference in female longevity between the two CO2 levels (Fig. 3-A).

Effects of CO2 and temperature on fecundity, e.g., number of eggs per female, were different from their ef-fects on longevity; fecundity was significantly influenced by both CO2 and temperature, but not by their interaction (Table). The brachypterous females deposited 48.1% more eggs in elevated temperature treatments than in ambient temperature treatments, and 36.1% more eggs in elevated CO2 treatments than in ambient CO2 treat-ments (Fig. 3-B).

Feeding amount by the planthopper

Honeydew excretion, which reflects the feeding amount of planthopper, was significantly affected by tempera-ture, but not by CO2 concentration and its interaction with temperature (Table). Honeydew excreted by the

brachypterous females decreased 28.7% in elevated temperature treatments than in ambient temperature treatments (Fig. 3-C).

DISCUSSION

Elevated CO2 increased rice stem height and biomass of stem parts and root. Elevated temperature enhanced rice stem height, leaf area, and biomass of stem parts, but reduced ratio of root to stem parts. These results are consistent with many other studies (e.g., Pritchard et al. 1999; Johns et al. 2003; Cheng et al. 2009). There were significant interactive effects between CO2 and temperature on leaf chlorophyll content and water con-tent of stem parts. Leaf chlorophyll content increased under elevated CO2, but only in ambient temperature

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Fig. 2 Effects of CO2 concentration and temperature on development of the brown planthopper on rice plants. A, nymphal duration (n=56-59). B, adult weight (n=40-45).

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Fig. 3 Effects of CO2 concentration and temperature on reproduction and feeding of the brown planthopper on rice plants. A, female longevity. B, number of eggs per female. C, honeydew excretion in 24 h per three brachypterous females. n=17-19 for each treatment.



treatment. This result is different from some reports of response of chlorophyll pigments to enhanced CO2 (Sallas et al. 2003), but is consistent with that in oilseed rape (Himanen et al. 2008), where chlorophyll concen-tration tended to increase in elevated CO2 under ambient temperature. Water content of stem parts was reduced by elevated temperature only in this study. Other stud-ies have provided contradictory results (Williams et al. 2000). In the study by Williams et al. (2000), leaf water content declined in red maple under elevated CO2, but was reduced in sugar maple under elevated temperature.

We found that development time from egg to adult was not affected by elevated CO2, but as expected, was accelerated by a 3°C rise in temperature, which may enhance population growth in the future climate scenario.

Insects feeding on foliage grown at elevated CO2 have frequently been shown to increase consumption and these results are generally explained as a response to reduced foliar quality, especially the reduction in leaf N (Bezemer and Jones 1998; Pritchard et al. 1999; Hughes and Bazzaz 2001; Goverde and Erhardt 2003). There are also many studies reporting little effect of CO2 level on folivorous insects (Bezemer et al. 1998; Johns et al. 2003; Mondor et al. 2010). Both nymph and adult of the brown planthopper suck phloem sap from leaf sheath, instead of leaf blade. We found no effects of CO2 level on feeding amount by the F1 brachypterous female adults. The reduced plant nutritional quality at elevated CO2 has been measured overwhelmingly to whole plant tissues (Chen et al. 2004; Flynn et al. 2006), but the sucking insects feed only on phloem sap, which may be of different nutritional quality from the whole tissues (Allen and Smith 1986). On the other hand, C/N ratios do not reflect other plant nutritional qualities which are key to sucking insects, such as the sucrose:amono acid ratio (Karley et al. 2002) and relative ratios of N, P, and K (Jansson and Ekbom 2002). This may explain why the leaf chewing insects show almost identical while the sucking insects show varying responses in feeding to elevated CO2. Elevated temperature reduced feeding amount of the F1 female adults by 28.7%, which confirms the results of Yu and Wu (1991) and Piyaphongkul (2013). The elevated temperature may directly act on the insects to reduce feeding amount, or the reduced water content in stem parts has altered osmotic potential in phloem-sap and reduced feeding by the planthopper

(Mattson and Haack 1987; Douglas 2006). Weight of the F1 adults was reduced under elevated temperature, which is in agreement with previous reports of other insects (Johns and Hughes 2002; Flynn et al. 2006; Himanen et al. 2008). A possible explanation for the reduced weight of the F1 adults may be that high tem-perature accelerated insect development and the nymphs did not feed sufficient nutrients; another reason may be that elevated temperature reduced feeding amount of the nymphs, as that in the F1 female adults, although we did not measure feeding amount of the nymphs.

Fecundity of the females was higher at both elevat-ed temperature and elevated CO2. Xiao et al. (2011) reported a significant increase in population size of the brown planthopper at elevated CO2. Other studies reported varying responses in fecundity of herbivores to elevated CO2, such as reduced fecundity in the aphid Phyllaphis fagi (Docherty et al. 1997), the mite Tetrany-chus urticae Koch (Joutei et al. 2000) and the pea aphid A. pisum (Mondor et al. 2010), unchanged fecundity in the Western flower thrips Frankliniella occidentalis (Heagle 2003), and increased fecundity in several other aphid species (Chen et al. 2004; Peltonen et al. 2006). Also, responses of herbivore population size to CO2 levels vary with host plant species. Population size of the aphid Macrosiphum euphorbiae increased on the C3 plant Solanum dulcamara while remains unchanged on the C4 plant Amaranthus viridis at elevated CO2 in comparison with ambient CO2 (Sudderth et al. 2005). The response of planthopper fecundity to elevated tem-perature in this study is consistent with those reported by Li (1984) and Shi et al. (2014). The brown plantho-pper occurs mainly in summer and autumn (Duck and Thomas 1979) and population size and severity of the pest depends to a great extent on a warming autumn (Cheng and Zhu 2006); on the other hand, increase in global-average surface temperatures is observed espe-cially in autumn (Busch et al. 2008). The elevated tem-perature in this study was a 3°C increase from 25/22 to 28/25°C (day/night cycle), which roughly corresponds to a warm autumn temperature and falls within the optimal temperature range for the brown planthopper (Shi et al. 2014), and therefore, it can be reasonably expected that fecundity of the brown planthopper increased at elevated temperature in this study. Under enriched CO2 atmo-sphere, nutritional value of host plants may deteriorate



due to dilution of N, but as in the pea aphid A. pisum, phloem sap suckers can actively elicit host responses to promote amino acid metabolism in both the host plant and in its bacteriocytes to favor its population growth under elevated CO2 (Guo et al. 2013).

CONCLUSION

The present study revealed significant effects of elevat-ed CO2 and/or elevated temperature on rice biological parameters, and a significant interaction between tem-perature and CO2 concentration on rice leaf chlorophyll content and water content of stem parts. Brown plan-thopper nymphal duration, adult weight and honeydew excretion was reduced under elevated temperatures, while female fecundity was higher at global change conditions. Adult longevity was affected by a significant interaction between CO2 and temperature. These results indicate interactive effects between temperature and CO2 on some biological parameters of both rice plant and the planthopper and also point to an increase in planthopper population size under climate change conditions. Further studies are needed to examine the mechanisms underly-ing the present results.

MATERIALS AND METHODS

Insects, plants and treatments

Stock culture of the brown planthopper originated from a sample collected from the Experimental Station of the China National Rice Research Institute (Fuyang, Zhejiang Province, China) in 2008. The insects were then reared on rice seedlings (variety TN1) in mesh cages in a greenhouse at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing.

Rice (variety TN1) was seeded in plastic pots (about 900 mL in volume, 10 cm in diameter, 12 cm in height) with a mixture of paddy soil, vermiculite and turfy soil (10:1:2) with pH=5.7, organic C=2.89%, total N=1.88 g kg-1, available P=3.22 mg kg-1, and available K=98.16 mg kg-1. The seedlings were incubated in climate chambers (HP400 GS-C, Wuhan Ruihua Equipment Co., Ltd., China) among four treatments: (1) ambient CO2 and temperature; (2) elevated CO2 and ambient temperature; (3) ambient CO2 and elevated temperature; and (4) elevated CO2 and temperature. The mean elevated CO2 concentration was approximately 700 ppm, and the ambient CO2 concentration approximately 360 ppm, which corresponds

to the predicted CO2 concentration at the end of the century and the current value (IPCC 2001), respectively. The elevated temperature was, on the average, +3°C higher than that in the control chambers, which was designed to correspond to the global warming scenarios predicted after a doubling of the atmospheric CO2 concentration (IPCC 2001). CO2 concentration in the chambers was controlled automatically by a sensor. Temperature was 28°C/25°C for day/night cycle for elevated temperature and 25°C/22°C, for ambient temperature, which was controlled automatically by a sensor. For the four experimental chambers, photoperiod was 14 h L:10 h D, with lights on at 7:00 a.m.; illumination intensity was 5 500-6 500 lx, and relative humidity was 70-80%. The plants were thinned on the 10th day after germination to leave only two hills per pot and watered as necessary. The four treatment batches were randomly re-assigned to new chambers weekly over the period when the experiment was conducted, and plant positions within each chamber rearranged at the same time. Although this procedure does not completely overcome the problem of pseudoreplication, it was designed to minimize any chamber effects as much as possible (Johns and Hughes 2002).

Rice plant development

To detect the treatment effects on rice plant development, biological parameters of 50-d-old rice plants were measured. Height of the primary stem was measured using a ruler. Leaf area and chlorophyll content of the reciprocal 2nd leaf on the primary stem were measured, using a leaf area meter YMJ-B (Zhejiang Top Instrument Company, China) and a chlorophyll meter SPAD 250Plus (Konica Minolta Sensing, Inc., Japan). And then, the rice plants were cut at the stem base and weighed for fresh weight of the aboveground parts. After being dried at 80°C for 72 h, the stem parts were weighed for dry weight. Roots of the rice plants were excavated from the pots and washed off mud in running water, and then weighed after being dried. Water content in the stem parts and ratio of root to stem parts were calculated.

Development of F1 generation of the planthopper

Development of F1 generation of the planthopper was observed in glass tubes (2.5 cm in diameter, 30 cm in height) placed in the four treatment chambers. Cohorts of planthoppers were obtained by confining one 4-d old, mated brachypterous female on a caged rice hill for oviposition for 24 h. The newly hatched nymphs (<24 h) were transferred singly to glass tubes, where there was a 45-d-old rice plant transplanted from the potted rice hills cultured in the four treatments. Rice aquaculture liquid was added to the glass tubes and the rice plants therein were replaced with new plants every 5 d. A slice of sponge was placed on the bottom of the glass tube to secure the rice plant and to prevent drowning of the planthopper. Nymphs



in the glass tubes were observed every 24 h for emergence. Emerged adults were sorted according to wing form and sex, and weighed using the electronic balance. Nymphal duration was calculated from the dates between hatching and adult emergence.

Longevity and fecundity of female adults of F1 generation

The treatment-mediated effects on fecundity of F1 generation were examined. One potted plant (45-50-d-old) was covered with a transparent, open-end plastic bottle (380 mL in volume). To secure the bottle, the low end of the bottle was inserted into soil in the pot; and to prevent insect escape, the bottle neck opening was sealed with sponge. The soil surface in the bottle was covered with sponge (0.5 cm in height) to prevent insect drowning. Thirty holes were punched evenly on the bottle wall with an insect pin to allow for air exchange and evaporation. One brachypterous female and one macropterous male within 24 h of emergence were paired in the above arena in the chambers of their original treatments. Six days since adult pairing, nymphs in the arena, if any, were recorded and then removed every day until there was no nymph hatching for 3 consecutive days. Survival of the female adult was also noticed, and female longevity was calculated from the emergence date and the death date.

Feeding amount by the planthopper

Honeydew excretion is an indicator of feeding amount of the planthopper (Pathak et al. 1982). Parafilm sachet method (Pathak et al. 1982) was employed to measure honeydew excretion. One sachet (3.3 cm×6.5 cm) was enwrapped to a primary rice stem at 5 cm above soil. Three brachypterous females (24-h-old, starved for 3 h) were transferred into a parafilm sachet using an insect aspirator. After feeding for 24 h, the females were removed and the sachet was weighed on an electronic balance (Al204, Mettler Toledo, to the nearest of 0.1 mg ). After the honeydew in the sachet was collected by a 50 μL microsyringe, the sachet was weighed again. The difference in the sachet weight was regarded as honeydew excretion. There might have been evaporation, but since both treatments were tested together, evaporation was assumed to be equivalent.

Statistical analysis

Data are expressed as means±SE. Data collected for each variable (except ratio of different groups of adults) were analyzed by a General Linear Model (GLM) (SPSS 1997), with CO2 concentration and temperature included as factors.

Datasets were transformed where necessary to satisfy assumptions of normality and homogeneity of variances (Day and Quinn 1989) for ANOVA and GLM. Data of honeydew excretion was natural-logarithms transformed, and water content of stem parts was square arcsine transformed. Where a significant interaction between the two factors occurred in the GLM analysis, an unplanned posthoc comparison of means was carried out (SPSS 1997). Levene’s test statistic was used prior to means comparisons to determine whether within-group variances differed significantly (P=0.05) (Day and Quinn 1989). For datasets with homogenous variances, Tukey’s HSD test was used (Day and Quinn 1989). Where heteroskedasticity occurred that could not be removed via data transformation, the Games and Howell method was used to compare means (Day and Quinn 1989). Chi-square test was used to compare the ratios of different groups of adults between treatments.

AcknowledgementsThe research was funded by the National Basic Research Program of China (2010CB951503).

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Interactive Effects of Elevated CO2 and Temperature on Rice Planthopper, Nilaparvata lugens