Subtropical Tritrophic Interactions Under Elevated CO2 and Temperature Conditions

Subtropical Tritrophic Interactions Under Elevated CO2 and Temperature Conditions Abstract The effects of climate change and extreme weather conditions on plants and animals have been documented extensively. However, the possible effects of these factors on plant–insect interactions in subtropical regions are relatively unexplored. The present study investigated the consequences of elevated CO2 and temperature on a tritrophic system (plant–insect–parasitoid) in subtropical regions. The experimental conditions were as follows: ambient CO2, 500 ppm; elevated CO2, 1,000 ppm; ambient temperature, 24/21°C (day/night); and elevated temperature, 29/26°C (day/night). Brassica oleracea var. italica foliar primary metabolites were quantified 6 wk after germination and insect feeding bioassays were subsequently conducted. Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae were fed directly on these plants until pupal development. In addition, the second instar S. litura larvae were exposed to the parasitoid Snellenius manilae (Ashmead) (Hymenoptera: Braconidae) under the same plant treatment conditions. The results suggested that elevated CO2 has a major influence on plant performance and foliar quality. Elevated CO2 also affected the leaf area, foliar fresh and dry weights, and total nitrogen and carbohydrate contents. Elevated temperature reduced the larval development time and increased the growth rate of S. litura. Sn. manilae had a higher parasitism rate and shorter development time at elevated temperature compared with ambient temperature. These results suggested that the dynamic and communal structure of S. litura and its parasitoids requires comprehensive evaluation in terms of the changes in nutritional quality (bottom-up control) caused by the interactive effects of CO2 and temperature. tritrophic system, elevated temperature, elevated CO2, climate change, natural enemy A tritrophic interaction can be defined as the ecological effects of three trophic levels, namely plants, herbivores, and their natural enemies, on one another. The interactions of these three levels have been extensively studied using plant-defensive and volatile chemicals (Heil 2008). In addition to plant-defensive chemicals, plant nutritional quality may affect the tritrophic interactions. Several studies have reported the effects of nutritional variation on insect development (Karley et al. 2002, Wetzel et al. 2016) and the performance of natural enemies (Hunter 2003, Gols et al. 2009). Therefore, plant nutritional quality has a crucial influence on the second and third trophic levels as the foundation of a tritrophic system. However, trophic-level interactions are inherently complex, even in the absence of climate change. In recent decades, temperature and atmospheric CO2 concentration have increased rapidly because of the increased concentration of greenhouse gases due to anthropogenic activities (Schuur et al. 2015, Hansen and Sato 2016). A report from the Intergovernmental Panel on Climate Change predicted that the global average surface temperature will increase by 1.4 to 5.8°C by 2100 and atmospheric CO2 concentration, which was 300 ppm in 1960, will reach 710 ppm by 2100 (Cubasch et al. 2013). Previous studies have demonstrated the negative effects of climate change on crop productivity (Lobell et al. 2011, Schauberger et al. 2017), as well as their influence on insect phenology (Van asch et al. 2007). One study revealed that butterflies in the northwest Mediterranean Basin underwent phenological changes because of elevated temperature (Stefanescu et al. 2003). In addition, elevated CO2 levels can directly affect photosynthesis and insect development; e.g., elevated CO2-related changes in plant quality altered the quality of aphids as a prey species for Leis axyridis (Pallas) (Coleoptera: Coccinellidae) through the food chain (Chen et al. 2005). Furthermore, elevated CO2 increased aphid survival and prolonged lady beetle development times (Gao et al. 2008). In reality, climate change is not the result of a single factor, and multiple factors might coexist simultaneously. Therefore, considering the effects of a single climate change factor might be inadequate for predicting the future effects of climate change on the ecosystem. Furthermore, the effects of climate change on plant–herbivore interactions have been documented extensively in recent years (DeLucia et al. 2012, Rosenblatt and Schmitz 2016, Lemoine et al. 2017). However, the possible effects of climate change on plant–insect–parasitoid interactions in tropical and subtropical regions are relatively unexplored. The present study investigated the effects of elevated CO2 and temperature on a tritrophic system (plant–insect–parasitoid) common in subtropical regions. The specific objectives of this study were to quantify the foliar primary metabolites of broccoli (Brassica oleracea var. italica) and evaluate the performance of the insect Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) and its parasitoid Snellenius manilae (Ashmead) (Hymenoptera: Braconidae) under the conditions of elevated temperature and CO2. Materials and Methods Greenhouse Conditions The responses of plants, herbivorous insects, and parasitoids to elevated temperature and CO2 were evaluated under controlled greenhouse conditions at National Chung Hsing University (Taichung, Taiwan) from November 2016 to January 2017. Four greenhouse rooms provided the following conditions: 1) ambient temperature of 24/21°C (day/night) with ambient CO2 (500 ppm), 2) ambient temperature of 24/21°C (day/night) with elevated CO2 (1,000 ppm), 3) elevated temperature of 29/26°C (day/night) with ambient CO2 (500 ppm), and 4) elevated temperature of 29/26°C (day/night) and elevated CO2 (1,000 ppm). A photoperiod of 12:12 (L:D) h was maintained for all treatments. The ambient temperature setting in this experiment was based on the average temperature of the local area in Taichung in each November during 2013–2016 (data from the Central Weather Bureau Taiwan), when broccoli plantations are most common in Taiwan. Sn. manilae is the most effective and common parasitoid of S. litura and is usually abundant in October and November. The elevated temperature setting was based on the predicted maximum temperature in the next century, which is expected to be at least +5°C (Cubasch et al. 2013). The ambient CO2 setting was based on the local ambient atmospheric CO2 concentration and the elevated CO2 setting was twice the ambient CO2 concentration (Long et al. 2004). The CO2 concentration in the greenshouse was first measured before the experiment and the concentration was found to be about 480 ± 20 ppm. Therefore, we set the ambient CO2 at 500 ppm. Plant Cultivation Conditions To examine the plant responses to elevated temperature and CO2, broccoli plant seeds were purchased from a local seed company (Known-You Seed Company, Kaohsiung, Taiwan). These seeds were soaked in warm water (45°C) for 30 min and rinsed with distilled water three times to accelerate germination. The seeds were then sown in standard potting soil (MOS-010; Known-You Seed Company) in 104-well plates and watered daily and maintained under ambient temperature and CO2 conditions. The seedlings with 1–2 true leaves were transplanted into plastic pots (10.5 [height] × 12 cm [diameter]) filled with standard soil and maintained in each of the four greenhouse rooms (treatments). Pseudoreplication would be very hard to avoid due to constraints imposed by the available facilities. Thus, we rearranged the pots randomly once a week to reduce the effects of within-chamber temperature and CO2 variation. The 42-d-old plants were collected for plant performance analysis. The leaf area, and biomass of aboveground parts were measured as indicators of plant growth performance (20 replicates per treatment, × 4 treatments = 80 plants). Insect Cultures S. litura eggs were collected from a field in Taichung County, Taiwan, and stored in a plastic rearing cup (250 ml) containing small moistened cotton sticks (7 [length] × 1 cm [diameter]). The hatched larvae were reared in a growth chamber at a constant temperature of 27 ± 2°C and 70 ± 3% relative humidity for 16:8 (L:D) h and fed with an artificial diet prepared as described by Gupta et al. (2005). The pupae were collected and individuals were sexed after reaching adulthood. Subsequently, the adult males and females were paired (10 pairs) in a glass cylinder (22 [height] × 14.5 cm [diameter]) lined with tissue paper for egg collection. The glass cylinder was maintained at room temperature and the paired adult insects were fed by saturated sugar solution (Yadav et al. 2010). One colony was maintained throughout this study. The S. litura larvae parasitized by Sn. manilae were collected from a vegetable field in Taichung County, Taiwan. The newly emerged female and male Sn. manilae adults were paired. Each pair was provided with several second and third instar S. litura larvae for oviposition, and fed with a sugar solution until the occurrence of larval development into cocoons, after which they were placed individually in small Petri dishes (1 [height] × 5.5 cm [diameter], Alpha Plus Scientific Co, Taoyuan, Taiwan). The insects were reared in the laboratory for several generations before use. Long-Term Insect Feeding Assay Long-term developmental trials were conducted to evaluate the effects of temperature and CO2 on plant growth and nutritional status, subsequently on S. litura development and growth throughout the larval feeding and pupal stages. The bioassay was performed after the eggs were hatched. The newly hatched larvae were weighed and each larva was individually transferred to a broccoli plant covered with a nylon net bag (35 × 35 × 45 cm) that allowed the insect to move freely (15 replicates per treatment, × 4 treatments = 60 larvae). Thereafter, the plants and insects were maintained under the different greenhouse conditions mentioned previously. At the pupal stage, each pupa was weighed 2 d after pupation and enclosed in a plastic rearing cup (250 ml) until reaching adulthood. The individual growth rate of each larva was calculated according to the method used by Gotthard et al. (1994): growth rate = (ln [pupal weight] − ln [hatching weight])/ larval time. The growth rate represents mean daily weight gain. Means and standard errors (SEs) were calculated for the initial neonate larval weight, the 2-d old pupal weight; larval and pupal development times; and the insect growth rate. The insects were weighed using Sartorius Micro Balance (Sartorius M2P, Goettingen, Germany). In the long-term feeding assay, additional leaf material from the test plants was collected during the bioassay for the measurement of foliar water and total nitrogen and carbohydrate contents. Parasitoid Development Assay The parasitoid development assay was conducted to examine parasitoid responses to elevated temperature and CO2. The first instar S. litura larvae were transferred to broccoli plants and covered with a nylon net bag (35 × 35 × 45 cm). After the larvae became second instar, 2-d-old mated female parasitoids were exposed to the larvae for 24 h in a nylon net bag containing water. Each cage contained one mated-female parasitoid and 40 larvae. This experiment was conducted with eight replicates for each treatment room (8 replicates per treatment, × 4 treatments = 32 cages). After 24 h exposure, the parasitoids were separated from the host larvae and the caterpillars were placed in a plastic rearing cup (250 ml). The caterpillars were then fed with broccoli leaves and maintained under various greenhouse conditions identical to those of the plant growth experiment described previously. The host larvae were monitored daily until pupal development. Parasitism was confirmed, if a parasitoid larva exited the host and initiated cocoon formation. The number of parasitized S. litura and parasitoid development time were recorded. Foliar Chemical Analysis To accompany the insect feeding studies, extra foliage (broccoli leaves similar to those used in the bioassays; 20 plants per treatment, × 4 treatments = 80 plants) was collected from the plants for foliar chemical analysis. The leaves were flash frozen in liquid nitrogen, freeze-dried, ground, and stored in a freezer at −20°C. Water, total nitrogen, and carbohydrate contents were quantified for each foliar sample and standard micro-Kjeldahl assays were conducted for leaf nitrogen quantification. The leaf samples were digested in acid (Parkinson and Allen 1975), and the aliquots were subjected to a Kjeltec auto system (Model 2300; Foss Tecator, Höganäs, Sweden) for nitrogen quantification. Glycine p-toluenesulfonate (Sigma Chemical Co., Steinheim, Germany) was used as standard. An enzymatic method was used to measure the total nonstructural carbohydrates (TNC; starch plus soluble carbohydrates) by using amyloglucosidase to hydrolyze starch before reducing the sugar assay (Madsen 1997). Statistical Analysis For the bioassays, the means are presented with SEs. The effects of elevated temperature and CO2 on the plant–insect–parasitoid interactions were examined through a two-way analysis of variance (ANOVA) with ‘temperature’ and ‘CO2’ and their interaction as factors, by using SAS for Windows (version 8, SAS Institute Inc, Cary, NC, 1999). The percentage of parasitism rates were transformed to arcsine square root values for the ANOVA. Tukey’s multiple range test was conducted to evaluate the differences between the mean values of each treatment. Results The plant performance results indicated that elevated CO2 (1,000 ppm) significantly enhanced plant biomass production, yielding increases in fresh weight, dry weight, and leaf area of 32, 57, and 48%, respectively, compared with the ambient CO2-treated plant (Table 1). By contrast, elevated temperature-treated plants had decreased plant biomass production and reductions in fresh weight, dry weight, and leaf area by 39, 14, and 10%, respectively, compared with the ambient temperature–treated plants. In addition, elevated temperature and CO2 had significant interactive effects on plant fresh weight and leaf dry weight per cm2 leaf area (Table 1), indicating that the foliar fresh weight and leaf dry weight per cm2 leaf area increased by CO2 enrichment would be affected by temperature. Table 1. Biomass of broccoli plants grown under different temperature and CO2 conditions Growth environment Dry weight (g) Fresh weight (g) Leaf area (cm2) Leaf dry weight per cm2 leaf area AT/AC 0.76 ± 0.063b 6.97 ± 0.573bc 217 ± 12c 0.00343 ± 0.00014a AT/EC 1.00 ± 0.071a 11.61 ± 0.814a 322 ± 18a 0.00306 ± 0.00031b ET/AC 0.54 ± 0.034c 6.11 ± 0.400c 196 ± 12c 0.00277 ± 0.00016b ET/EC 0.92 ± 0.041a 8.14 ± 0.411b 263 ± 10b 0.00351 ± 0.00024a (df) F-values, P-values  Temperature (3,76) 7.35, 0.0089 (3,76) 13.95, 0.0004 (3,76) 9.32, 0.0035 (3,76) 0.78, 0.3799  CO2 (3,76) 33.34, <0.0001 (3,76) 33.17, <0.0001 (3,76) 41.88, <0.0001 (3,76) 2.76, 0.1021  Temperature × CO2 (3,76) 1.74, 0.1931 (3,76) 5.10, 0.0278 (3,76) 2.06, 0.1571 (3,76) 24.02, <0.0001 Growth environment Dry weight (g) Fresh weight (g) Leaf area (cm2) Leaf dry weight per cm2 leaf area AT/AC 0.76 ± 0.063b 6.97 ± 0.573bc 217 ± 12c 0.00343 ± 0.00014a AT/EC 1.00 ± 0.071a 11.61 ± 0.814a 322 ± 18a 0.00306 ± 0.00031b ET/AC 0.54 ± 0.034c 6.11 ± 0.400c 196 ± 12c 0.00277 ± 0.00016b ET/EC 0.92 ± 0.041a 8.14 ± 0.411b 263 ± 10b 0.00351 ± 0.00024a (df) F-values, P-values  Temperature (3,76) 7.35, 0.0089 (3,76) 13.95, 0.0004 (3,76) 9.32, 0.0035 (3,76) 0.78, 0.3799  CO2 (3,76) 33.34, <0.0001 (3,76) 33.17, <0.0001 (3,76) 41.88, <0.0001 (3,76) 2.76, 0.1021  Temperature × CO2 (3,76) 1.74, 0.1931 (3,76) 5.10, 0.0278 (3,76) 2.06, 0.1571 (3,76) 24.02, <0.0001 AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Dry weight, fresh weight, and leaf area (means ± SE; N = 20 plants per treatment). Statistical analysis was performed with a two-way ANOVA. Numbers with the same lowercase are not significantly different (P > 0.05). View Large Table 1. Biomass of broccoli plants grown under different temperature and CO2 conditions Growth environment Dry weight (g) Fresh weight (g) Leaf area (cm2) Leaf dry weight per cm2 leaf area AT/AC 0.76 ± 0.063b 6.97 ± 0.573bc 217 ± 12c 0.00343 ± 0.00014a AT/EC 1.00 ± 0.071a 11.61 ± 0.814a 322 ± 18a 0.00306 ± 0.00031b ET/AC 0.54 ± 0.034c 6.11 ± 0.400c 196 ± 12c 0.00277 ± 0.00016b ET/EC 0.92 ± 0.041a 8.14 ± 0.411b 263 ± 10b 0.00351 ± 0.00024a (df) F-values, P-values  Temperature (3,76) 7.35, 0.0089 (3,76) 13.95, 0.0004 (3,76) 9.32, 0.0035 (3,76) 0.78, 0.3799  CO2 (3,76) 33.34, <0.0001 (3,76) 33.17, <0.0001 (3,76) 41.88, <0.0001 (3,76) 2.76, 0.1021  Temperature × CO2 (3,76) 1.74, 0.1931 (3,76) 5.10, 0.0278 (3,76) 2.06, 0.1571 (3,76) 24.02, <0.0001 Growth environment Dry weight (g) Fresh weight (g) Leaf area (cm2) Leaf dry weight per cm2 leaf area AT/AC 0.76 ± 0.063b 6.97 ± 0.573bc 217 ± 12c 0.00343 ± 0.00014a AT/EC 1.00 ± 0.071a 11.61 ± 0.814a 322 ± 18a 0.00306 ± 0.00031b ET/AC 0.54 ± 0.034c 6.11 ± 0.400c 196 ± 12c 0.00277 ± 0.00016b ET/EC 0.92 ± 0.041a 8.14 ± 0.411b 263 ± 10b 0.00351 ± 0.00024a (df) F-values, P-values  Temperature (3,76) 7.35, 0.0089 (3,76) 13.95, 0.0004 (3,76) 9.32, 0.0035 (3,76) 0.78, 0.3799  CO2 (3,76) 33.34, <0.0001 (3,76) 33.17, <0.0001 (3,76) 41.88, <0.0001 (3,76) 2.76, 0.1021  Temperature × CO2 (3,76) 1.74, 0.1931 (3,76) 5.10, 0.0278 (3,76) 2.06, 0.1571 (3,76) 24.02, <0.0001 AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Dry weight, fresh weight, and leaf area (means ± SE; N = 20 plants per treatment). Statistical analysis was performed with a two-way ANOVA. Numbers with the same lowercase are not significantly different (P > 0.05). View Large Regarding foliar quality, elevated CO2 resulted in an increase of carbon to nitrogen (C/N) ratio and total nonstructural carbohydrate (TNC); elevated CO2 doubled C/N ratio at ambient temperature and increased by about 30% at elevated temperature. Similarly, elevated CO2 at ambient temperature and elevated temperature increased TNC from 20 to 30% which is 50% increase. On the other hand, elevated CO2 significantly reduced the nitrogen contentat at ambient temperature (0.835%) and elevated temperature (1.094%) (Table 2). Although elevated temperature yielded a slight increase in nitrogen content, it exerted less of an effect on plant nutrition quality compared with elevated CO2. Moreover, temperature and CO2 did not exert significant interactive effects on nitrogen content, TNC and C/N ratio (Table 2). Table 2. Foliar quality of broccoli plants grown under different temperature and CO2 conditions Growth environment Nitrogen content (%) Total nonstructural carbohydrate (%) C/N ratio AT/AC 1.574 ± 0.177b 21.73 ± 0.38c 14.94 ± 1.50b AT/EC 0.835 ± 0.100c 31.22 ± 0.62ab 30.81 ± 1.8a ET/AC 1.978 ± 0.116a 26.57 ± 0.39bc 18.07 ± 1.7b ET/EC 1.094 ± 0.221bc 35.49 ± 1.11a 23.57 ± 0.6ab (df) F-values, P-values  Temperature (3,76) 12.66, 0.0098 (3,76) 2.00, 0.1728 (3,76) 1.30, 0.2724  CO2 (3,76) 26.29, <0.0001 (3,76) 15.66, 0.0008 (3,76) 12.41, 0.0031  Temperature × CO2 (3,76) 0.66, 0.3723 (3,76) 2.44, 0.1338 (3,76) 1.80, 0.1931 Growth environment Nitrogen content (%) Total nonstructural carbohydrate (%) C/N ratio AT/AC 1.574 ± 0.177b 21.73 ± 0.38c 14.94 ± 1.50b AT/EC 0.835 ± 0.100c 31.22 ± 0.62ab 30.81 ± 1.8a ET/AC 1.978 ± 0.116a 26.57 ± 0.39bc 18.07 ± 1.7b ET/EC 1.094 ± 0.221bc 35.49 ± 1.11a 23.57 ± 0.6ab (df) F-values, P-values  Temperature (3,76) 12.66, 0.0098 (3,76) 2.00, 0.1728 (3,76) 1.30, 0.2724  CO2 (3,76) 26.29, <0.0001 (3,76) 15.66, 0.0008 (3,76) 12.41, 0.0031  Temperature × CO2 (3,76) 0.66, 0.3723 (3,76) 2.44, 0.1338 (3,76) 1.80, 0.1931 AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Nitrogen, TNC, C/N ratio (means ± SE; N = 20 plants per treatment). Statistical analysis was performed with a two-way ANOVA. Numbers with the same lowercase are not significantly different (P > 0.05). View Large Table 2. Foliar quality of broccoli plants grown under different temperature and CO2 conditions Growth environment Nitrogen content (%) Total nonstructural carbohydrate (%) C/N ratio AT/AC 1.574 ± 0.177b 21.73 ± 0.38c 14.94 ± 1.50b AT/EC 0.835 ± 0.100c 31.22 ± 0.62ab 30.81 ± 1.8a ET/AC 1.978 ± 0.116a 26.57 ± 0.39bc 18.07 ± 1.7b ET/EC 1.094 ± 0.221bc 35.49 ± 1.11a 23.57 ± 0.6ab (df) F-values, P-values  Temperature (3,76) 12.66, 0.0098 (3,76) 2.00, 0.1728 (3,76) 1.30, 0.2724  CO2 (3,76) 26.29, <0.0001 (3,76) 15.66, 0.0008 (3,76) 12.41, 0.0031  Temperature × CO2 (3,76) 0.66, 0.3723 (3,76) 2.44, 0.1338 (3,76) 1.80, 0.1931 Growth environment Nitrogen content (%) Total nonstructural carbohydrate (%) C/N ratio AT/AC 1.574 ± 0.177b 21.73 ± 0.38c 14.94 ± 1.50b AT/EC 0.835 ± 0.100c 31.22 ± 0.62ab 30.81 ± 1.8a ET/AC 1.978 ± 0.116a 26.57 ± 0.39bc 18.07 ± 1.7b ET/EC 1.094 ± 0.221bc 35.49 ± 1.11a 23.57 ± 0.6ab (df) F-values, P-values  Temperature (3,76) 12.66, 0.0098 (3,76) 2.00, 0.1728 (3,76) 1.30, 0.2724  CO2 (3,76) 26.29, <0.0001 (3,76) 15.66, 0.0008 (3,76) 12.41, 0.0031  Temperature × CO2 (3,76) 0.66, 0.3723 (3,76) 2.44, 0.1338 (3,76) 1.80, 0.1931 AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Nitrogen, TNC, C/N ratio (means ± SE; N = 20 plants per treatment). Statistical analysis was performed with a two-way ANOVA. Numbers with the same lowercase are not significantly different (P > 0.05). View Large Figure 1 shows the development of S. litura from larval stage to pupal stage under different temperature and CO2 conditions. The results demonstrated that S. litura larvae, which fed on broccoli plants grown at elevated temperature and CO2 concentration, had considerably changed development time and growth rate. Elevated CO2 prolonged the insect development, while elevated temperature shortened the insect development, However, combined elevated CO2 and temperature treatment, CO2 did not affect the insect development. Insect growth rate increased when grown under elevated temperature and the growth rate was higher when grown at combining elevated temperature and CO2 treatment. Fig. 1. View largeDownload slide Development of S. litura. Development time from the first instar to pupal stage, pupal weight, and growth rate of S. litura reared and fed on broccoli leaves grown under different temperature and CO2 conditions (N = 15). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Fig. 1. View largeDownload slide Development of S. litura. Development time from the first instar to pupal stage, pupal weight, and growth rate of S. litura reared and fed on broccoli leaves grown under different temperature and CO2 conditions (N = 15). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Larval development was shorter by 5–6 d and the growth rate was significantly increased by 15% at elevated temperature compared with the corresponding measurements under ambient temperature conditions. In addition, elevated CO2 significantly reduced the pupal weight by more than 50 mg, compared with the ambient CO2 treatment. Furthermore, temperature and CO2 had significant interactive effects on the S. litura larval development time and growth rate (Fig. 1). The results of Sn. manilae performance revealed that elevated temperature and CO2 would affect Sn. manilae development. At an elevated temperature, the egg–larval and cocoon development times decreased by 11–12 d and 2–3 d, respectively (Fig. 2). Moreover, elevated temperature significantly increased the cocoon weight and parasitism rate. In addition, elevated CO2 significantly reduced the egg–larval development time by nearly 5 d. Temperature and CO2 exerted significant interactive effects on egg–larval development time (Figs. 2 and 3). Fig. 2. View largeDownload slide Development of Sn. manilae. Egg–larval and cocoon development times and cocoon weight under different temperature and CO2 conditions (N = 30). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Fig. 2. View largeDownload slide Development of Sn. manilae. Egg–larval and cocoon development times and cocoon weight under different temperature and CO2 conditions (N = 30). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Fig. 3. View largeDownload slide Parasitism rate of Sn. manilae under different temperature and CO2 conditions (N = 8). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Fig. 3. View largeDownload slide Parasitism rate of Sn. manilae under different temperature and CO2 conditions (N = 8). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Discussion The present study demonstrated the effects of elevated temperature and CO2 on a tritrophic system involving broccoli, S. litura and Sn. manilae. Elevated CO2 mainly alters the biomass production and nitrogen content, TNC and C/N ratio of broccoli. The bioassay results reveal that the S. litura fed on elevated CO2-treated broccoli leaves exhibited negative performance with a longer larval development time and decreased pupal weight. The longer larval development times of the host typically related with reduced leaf quality may also extend window time for parasitoids to attack host (Stiling and Cornelissen, 2007). In addition, elevated temperature can directly stimulate the growth and development of S. litura and their parasitoid Sn. manilae likely due to an accelerated metabolism (Kooijman 2014, Akbar et al. 2016). The broccoli plants grown under elevated CO2 (1,000 ppm) conditions exhibited increased aboveground biomass and enlarged leaf area of almost 50% compared with those grown under ambient CO2 conditions. Similarly, a study by Pandey (2017) revealed that a high CO2 concentration can stimulate underground and aboveground biomass in wheat (Triticum aestivum L. var Kundan). However, the results of the present study demonstrate that elevated temperature reduces the growth-stimulating effects of CO2 on broccoli biomass, thereby suggesting that broccoli might be a thermosensitive plant that requires cooler temperatures to achieve optimal growth. Therefore, an increase in temperature of up to 5°C could substantially reduce broccoli biomass. Ciancaleoni (2016) studied broccoli yield response to environmental factors and reported that broccoli yield is largely determined by climate conditions during the growing season and deviations from optimal conditions can seriously affect yield. Nitrogen and carbohydrates are necessary for the growth and development of plant tissue (Uchida 2000). In the present study, the broccoli plants grown under elevated CO2 conditions had a lower total nitrogen content, whereas their TNC concentration (starch and sugar) increased by nearly 10%. Furthermore, our results demonstrate that the C/N ratio was higher in the elevated CO2-treated plants than in the ambient CO2-treated plants. The plant nutrition quality results are in accordance with several climate change projections (Reich et al. 2014, Sardans et al. 2017). Previous studies have reported that elevated CO2 can stimulate photosynthesis, reduce nitrogen concentration, and lead to an increased C/N ratio (Long et al. 2004, Ainsworth and Rogers 2007, De Souza et al. 2008, Leakey et al. 2009). The TNC concentration in plant tissue is considered an indicator of the overall carbon availability status, whereas the caloric value of plants is considered less vital for insect development because carbohydrates can be synthesized from fats by insects (Behmer 2009). However, nitrogen is essential for insect development, and thus foliage with decreased nitrogen concentration might be considered a poor food source for insects (Caulfield and Bunce 1994). Nevertheless, our results demonstrate that elevated temperature has less of an effect on plant nutrition quality under elevated CO2 conditions. The results of the present study clearly demonstrate that CO2 concentration is a crucial factor influencing herbivore and parasitoid performance. The S. litura fed on elevated CO2-treated broccoli leaves exhibited prolonged larval development time and decreased pupal weight, thereby showing consistency with the findings of previous studies, which have demonstrated that elevated CO2 exerts an indirect effect on insect growth and development by reducing the nutrition quality of the host plant (Srinivasa Rao et al. 2012). Interestingly, our study found that elevated CO2 prolonged larval development time of S. litura, in contrast elevated CO2 shortened larval development time of Sn. manilae. One possible explanation may be that the elevated CO2 can either increase or decrease production of volatile emissions and plant defensive compounds that can affect the parasitoid development (Heil 2008). For example, the study by Klaiber (2013) indicated that Brassica plants grown under elevated CO2 contained higher levels of glucosinolates which could reduce the body mass of aphids and shorten the parasitoid development time. In addition, one previous study suggested that the improved performance of the parasitoids Spilichneumon limnophilus (Thomson) (Hymenoptera: Ichneumonidae) and Chasmias paludator (Desvignes) (Hymenoptera: Ichneumonidae) is strongly correlated with body size, which is associated with plant vigor (Teder and Tammaru 2002). Our results reveal that although elevated temperature (29/26°C, day/night) has less of an effect on plant nutrition quality than does elevated CO2, it strongly affects insect development. Elevated temperature can increase the larval development time of S. litura. These findings are consistent with those of previous studies. One study reported that elevated temperature hinders S. litura development (Rao et al. 2014). The performance of the parasitoid Sn. manilae and its host S. litura show similar trends, both exhibiting decreased larval and cocoon development times. Moreover, at elevated temperature, the female parasitoids became more physically active, resulting in an increase (<15%) in the parasitism rate. The increased development times of the herbivorous host and increased parasitoid activity at elevated temperature in our study could be explained by Neven (2000), who reported that insect body temperature increases at high temperature with concomitant increases in insect metabolism and respiration, leading to changes in insect development and behaviors. Similarly, previous studies have revealed that Aenasius arizonensis (Girault) (Hymenoptera: Encyrtidae), an endoparasitoid of Phenacoccus solenopsis (Tinsley) (Hemiptera: Pseudococcidae), has the highest parasitism and emergence rates at 31°C (He et al. 2017). In addition, Sn. manilae has improved performance at elevated temperature, which consequently increases the cocoon weight and parasitism rate. These findings are consistent with results of numerous studies that have suggested parasitoids can develop under various thermal conditions because of their mating and host-locating behaviors (Stireman et al. 2005, Thomson et al. 2010). Parasitoids are more likely to encounter variable climate conditions and might have higher potential for adaptability than their herbivorous hosts, particularly in subtropical regions with average temperatures of 20–35°C. In conclusion, elevated temperature and CO2 significantly influenced tritrophic systems possibly by altering host plant nutrient availability, which further affects the growth and development of S. litura and Sn. manilae. We suggest that the dynamic and communal structure of S. litura and its parasitoids must be comprehensively evaluated in terms of the changes in their nutrient quality (bottom-up controlled) caused by variable CO2 and temperature conditions. Our study results could facilitate understanding of the interactions of trophic levels in future environments, particularly in subtropical regions. Acknowledgments The study was supported by grant from Ministry of Science and Technology (MOST), Taiwan (105-2313-B-005-003-MY3). We thank Wallace Academic Editing, Sharon Bailey and Mark Darvill. References Cited Ainsworth , E. A. , and A. Rogers . 2007 . The response of photosynthesis and stomatal conductance to rising [CO2]: mechanisms and environmental interactions . Plant. 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For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Entomology Oxford University Press

Subtropical Tritrophic Interactions Under Elevated CO2 and Temperature Conditions

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Abstract

Abstract The effects of climate change and extreme weather conditions on plants and animals have been documented extensively. However, the possible effects of these factors on plant–insect interactions in subtropical regions are relatively unexplored. The present study investigated the consequences of elevated CO2 and temperature on a tritrophic system (plant–insect–parasitoid) in subtropical regions. The experimental conditions were as follows: ambient CO2, 500 ppm; elevated CO2, 1,000 ppm; ambient temperature, 24/21°C (day/night); and elevated temperature, 29/26°C (day/night). Brassica oleracea var. italica foliar primary metabolites were quantified 6 wk after germination and insect feeding bioassays were subsequently conducted. Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae were fed directly on these plants until pupal development. In addition, the second instar S. litura larvae were exposed to the parasitoid Snellenius manilae (Ashmead) (Hymenoptera: Braconidae) under the same plant treatment conditions. The results suggested that elevated CO2 has a major influence on plant performance and foliar quality. Elevated CO2 also affected the leaf area, foliar fresh and dry weights, and total nitrogen and carbohydrate contents. Elevated temperature reduced the larval development time and increased the growth rate of S. litura. Sn. manilae had a higher parasitism rate and shorter development time at elevated temperature compared with ambient temperature. These results suggested that the dynamic and communal structure of S. litura and its parasitoids requires comprehensive evaluation in terms of the changes in nutritional quality (bottom-up control) caused by the interactive effects of CO2 and temperature. tritrophic system, elevated temperature, elevated CO2, climate change, natural enemy A tritrophic interaction can be defined as the ecological effects of three trophic levels, namely plants, herbivores, and their natural enemies, on one another. The interactions of these three levels have been extensively studied using plant-defensive and volatile chemicals (Heil 2008). In addition to plant-defensive chemicals, plant nutritional quality may affect the tritrophic interactions. Several studies have reported the effects of nutritional variation on insect development (Karley et al. 2002, Wetzel et al. 2016) and the performance of natural enemies (Hunter 2003, Gols et al. 2009). Therefore, plant nutritional quality has a crucial influence on the second and third trophic levels as the foundation of a tritrophic system. However, trophic-level interactions are inherently complex, even in the absence of climate change. In recent decades, temperature and atmospheric CO2 concentration have increased rapidly because of the increased concentration of greenhouse gases due to anthropogenic activities (Schuur et al. 2015, Hansen and Sato 2016). A report from the Intergovernmental Panel on Climate Change predicted that the global average surface temperature will increase by 1.4 to 5.8°C by 2100 and atmospheric CO2 concentration, which was 300 ppm in 1960, will reach 710 ppm by 2100 (Cubasch et al. 2013). Previous studies have demonstrated the negative effects of climate change on crop productivity (Lobell et al. 2011, Schauberger et al. 2017), as well as their influence on insect phenology (Van asch et al. 2007). One study revealed that butterflies in the northwest Mediterranean Basin underwent phenological changes because of elevated temperature (Stefanescu et al. 2003). In addition, elevated CO2 levels can directly affect photosynthesis and insect development; e.g., elevated CO2-related changes in plant quality altered the quality of aphids as a prey species for Leis axyridis (Pallas) (Coleoptera: Coccinellidae) through the food chain (Chen et al. 2005). Furthermore, elevated CO2 increased aphid survival and prolonged lady beetle development times (Gao et al. 2008). In reality, climate change is not the result of a single factor, and multiple factors might coexist simultaneously. Therefore, considering the effects of a single climate change factor might be inadequate for predicting the future effects of climate change on the ecosystem. Furthermore, the effects of climate change on plant–herbivore interactions have been documented extensively in recent years (DeLucia et al. 2012, Rosenblatt and Schmitz 2016, Lemoine et al. 2017). However, the possible effects of climate change on plant–insect–parasitoid interactions in tropical and subtropical regions are relatively unexplored. The present study investigated the effects of elevated CO2 and temperature on a tritrophic system (plant–insect–parasitoid) common in subtropical regions. The specific objectives of this study were to quantify the foliar primary metabolites of broccoli (Brassica oleracea var. italica) and evaluate the performance of the insect Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) and its parasitoid Snellenius manilae (Ashmead) (Hymenoptera: Braconidae) under the conditions of elevated temperature and CO2. Materials and Methods Greenhouse Conditions The responses of plants, herbivorous insects, and parasitoids to elevated temperature and CO2 were evaluated under controlled greenhouse conditions at National Chung Hsing University (Taichung, Taiwan) from November 2016 to January 2017. Four greenhouse rooms provided the following conditions: 1) ambient temperature of 24/21°C (day/night) with ambient CO2 (500 ppm), 2) ambient temperature of 24/21°C (day/night) with elevated CO2 (1,000 ppm), 3) elevated temperature of 29/26°C (day/night) with ambient CO2 (500 ppm), and 4) elevated temperature of 29/26°C (day/night) and elevated CO2 (1,000 ppm). A photoperiod of 12:12 (L:D) h was maintained for all treatments. The ambient temperature setting in this experiment was based on the average temperature of the local area in Taichung in each November during 2013–2016 (data from the Central Weather Bureau Taiwan), when broccoli plantations are most common in Taiwan. Sn. manilae is the most effective and common parasitoid of S. litura and is usually abundant in October and November. The elevated temperature setting was based on the predicted maximum temperature in the next century, which is expected to be at least +5°C (Cubasch et al. 2013). The ambient CO2 setting was based on the local ambient atmospheric CO2 concentration and the elevated CO2 setting was twice the ambient CO2 concentration (Long et al. 2004). The CO2 concentration in the greenshouse was first measured before the experiment and the concentration was found to be about 480 ± 20 ppm. Therefore, we set the ambient CO2 at 500 ppm. Plant Cultivation Conditions To examine the plant responses to elevated temperature and CO2, broccoli plant seeds were purchased from a local seed company (Known-You Seed Company, Kaohsiung, Taiwan). These seeds were soaked in warm water (45°C) for 30 min and rinsed with distilled water three times to accelerate germination. The seeds were then sown in standard potting soil (MOS-010; Known-You Seed Company) in 104-well plates and watered daily and maintained under ambient temperature and CO2 conditions. The seedlings with 1–2 true leaves were transplanted into plastic pots (10.5 [height] × 12 cm [diameter]) filled with standard soil and maintained in each of the four greenhouse rooms (treatments). Pseudoreplication would be very hard to avoid due to constraints imposed by the available facilities. Thus, we rearranged the pots randomly once a week to reduce the effects of within-chamber temperature and CO2 variation. The 42-d-old plants were collected for plant performance analysis. The leaf area, and biomass of aboveground parts were measured as indicators of plant growth performance (20 replicates per treatment, × 4 treatments = 80 plants). Insect Cultures S. litura eggs were collected from a field in Taichung County, Taiwan, and stored in a plastic rearing cup (250 ml) containing small moistened cotton sticks (7 [length] × 1 cm [diameter]). The hatched larvae were reared in a growth chamber at a constant temperature of 27 ± 2°C and 70 ± 3% relative humidity for 16:8 (L:D) h and fed with an artificial diet prepared as described by Gupta et al. (2005). The pupae were collected and individuals were sexed after reaching adulthood. Subsequently, the adult males and females were paired (10 pairs) in a glass cylinder (22 [height] × 14.5 cm [diameter]) lined with tissue paper for egg collection. The glass cylinder was maintained at room temperature and the paired adult insects were fed by saturated sugar solution (Yadav et al. 2010). One colony was maintained throughout this study. The S. litura larvae parasitized by Sn. manilae were collected from a vegetable field in Taichung County, Taiwan. The newly emerged female and male Sn. manilae adults were paired. Each pair was provided with several second and third instar S. litura larvae for oviposition, and fed with a sugar solution until the occurrence of larval development into cocoons, after which they were placed individually in small Petri dishes (1 [height] × 5.5 cm [diameter], Alpha Plus Scientific Co, Taoyuan, Taiwan). The insects were reared in the laboratory for several generations before use. Long-Term Insect Feeding Assay Long-term developmental trials were conducted to evaluate the effects of temperature and CO2 on plant growth and nutritional status, subsequently on S. litura development and growth throughout the larval feeding and pupal stages. The bioassay was performed after the eggs were hatched. The newly hatched larvae were weighed and each larva was individually transferred to a broccoli plant covered with a nylon net bag (35 × 35 × 45 cm) that allowed the insect to move freely (15 replicates per treatment, × 4 treatments = 60 larvae). Thereafter, the plants and insects were maintained under the different greenhouse conditions mentioned previously. At the pupal stage, each pupa was weighed 2 d after pupation and enclosed in a plastic rearing cup (250 ml) until reaching adulthood. The individual growth rate of each larva was calculated according to the method used by Gotthard et al. (1994): growth rate = (ln [pupal weight] − ln [hatching weight])/ larval time. The growth rate represents mean daily weight gain. Means and standard errors (SEs) were calculated for the initial neonate larval weight, the 2-d old pupal weight; larval and pupal development times; and the insect growth rate. The insects were weighed using Sartorius Micro Balance (Sartorius M2P, Goettingen, Germany). In the long-term feeding assay, additional leaf material from the test plants was collected during the bioassay for the measurement of foliar water and total nitrogen and carbohydrate contents. Parasitoid Development Assay The parasitoid development assay was conducted to examine parasitoid responses to elevated temperature and CO2. The first instar S. litura larvae were transferred to broccoli plants and covered with a nylon net bag (35 × 35 × 45 cm). After the larvae became second instar, 2-d-old mated female parasitoids were exposed to the larvae for 24 h in a nylon net bag containing water. Each cage contained one mated-female parasitoid and 40 larvae. This experiment was conducted with eight replicates for each treatment room (8 replicates per treatment, × 4 treatments = 32 cages). After 24 h exposure, the parasitoids were separated from the host larvae and the caterpillars were placed in a plastic rearing cup (250 ml). The caterpillars were then fed with broccoli leaves and maintained under various greenhouse conditions identical to those of the plant growth experiment described previously. The host larvae were monitored daily until pupal development. Parasitism was confirmed, if a parasitoid larva exited the host and initiated cocoon formation. The number of parasitized S. litura and parasitoid development time were recorded. Foliar Chemical Analysis To accompany the insect feeding studies, extra foliage (broccoli leaves similar to those used in the bioassays; 20 plants per treatment, × 4 treatments = 80 plants) was collected from the plants for foliar chemical analysis. The leaves were flash frozen in liquid nitrogen, freeze-dried, ground, and stored in a freezer at −20°C. Water, total nitrogen, and carbohydrate contents were quantified for each foliar sample and standard micro-Kjeldahl assays were conducted for leaf nitrogen quantification. The leaf samples were digested in acid (Parkinson and Allen 1975), and the aliquots were subjected to a Kjeltec auto system (Model 2300; Foss Tecator, Höganäs, Sweden) for nitrogen quantification. Glycine p-toluenesulfonate (Sigma Chemical Co., Steinheim, Germany) was used as standard. An enzymatic method was used to measure the total nonstructural carbohydrates (TNC; starch plus soluble carbohydrates) by using amyloglucosidase to hydrolyze starch before reducing the sugar assay (Madsen 1997). Statistical Analysis For the bioassays, the means are presented with SEs. The effects of elevated temperature and CO2 on the plant–insect–parasitoid interactions were examined through a two-way analysis of variance (ANOVA) with ‘temperature’ and ‘CO2’ and their interaction as factors, by using SAS for Windows (version 8, SAS Institute Inc, Cary, NC, 1999). The percentage of parasitism rates were transformed to arcsine square root values for the ANOVA. Tukey’s multiple range test was conducted to evaluate the differences between the mean values of each treatment. Results The plant performance results indicated that elevated CO2 (1,000 ppm) significantly enhanced plant biomass production, yielding increases in fresh weight, dry weight, and leaf area of 32, 57, and 48%, respectively, compared with the ambient CO2-treated plant (Table 1). By contrast, elevated temperature-treated plants had decreased plant biomass production and reductions in fresh weight, dry weight, and leaf area by 39, 14, and 10%, respectively, compared with the ambient temperature–treated plants. In addition, elevated temperature and CO2 had significant interactive effects on plant fresh weight and leaf dry weight per cm2 leaf area (Table 1), indicating that the foliar fresh weight and leaf dry weight per cm2 leaf area increased by CO2 enrichment would be affected by temperature. Table 1. Biomass of broccoli plants grown under different temperature and CO2 conditions Growth environment Dry weight (g) Fresh weight (g) Leaf area (cm2) Leaf dry weight per cm2 leaf area AT/AC 0.76 ± 0.063b 6.97 ± 0.573bc 217 ± 12c 0.00343 ± 0.00014a AT/EC 1.00 ± 0.071a 11.61 ± 0.814a 322 ± 18a 0.00306 ± 0.00031b ET/AC 0.54 ± 0.034c 6.11 ± 0.400c 196 ± 12c 0.00277 ± 0.00016b ET/EC 0.92 ± 0.041a 8.14 ± 0.411b 263 ± 10b 0.00351 ± 0.00024a (df) F-values, P-values  Temperature (3,76) 7.35, 0.0089 (3,76) 13.95, 0.0004 (3,76) 9.32, 0.0035 (3,76) 0.78, 0.3799  CO2 (3,76) 33.34, <0.0001 (3,76) 33.17, <0.0001 (3,76) 41.88, <0.0001 (3,76) 2.76, 0.1021  Temperature × CO2 (3,76) 1.74, 0.1931 (3,76) 5.10, 0.0278 (3,76) 2.06, 0.1571 (3,76) 24.02, <0.0001 Growth environment Dry weight (g) Fresh weight (g) Leaf area (cm2) Leaf dry weight per cm2 leaf area AT/AC 0.76 ± 0.063b 6.97 ± 0.573bc 217 ± 12c 0.00343 ± 0.00014a AT/EC 1.00 ± 0.071a 11.61 ± 0.814a 322 ± 18a 0.00306 ± 0.00031b ET/AC 0.54 ± 0.034c 6.11 ± 0.400c 196 ± 12c 0.00277 ± 0.00016b ET/EC 0.92 ± 0.041a 8.14 ± 0.411b 263 ± 10b 0.00351 ± 0.00024a (df) F-values, P-values  Temperature (3,76) 7.35, 0.0089 (3,76) 13.95, 0.0004 (3,76) 9.32, 0.0035 (3,76) 0.78, 0.3799  CO2 (3,76) 33.34, <0.0001 (3,76) 33.17, <0.0001 (3,76) 41.88, <0.0001 (3,76) 2.76, 0.1021  Temperature × CO2 (3,76) 1.74, 0.1931 (3,76) 5.10, 0.0278 (3,76) 2.06, 0.1571 (3,76) 24.02, <0.0001 AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Dry weight, fresh weight, and leaf area (means ± SE; N = 20 plants per treatment). Statistical analysis was performed with a two-way ANOVA. Numbers with the same lowercase are not significantly different (P > 0.05). View Large Table 1. Biomass of broccoli plants grown under different temperature and CO2 conditions Growth environment Dry weight (g) Fresh weight (g) Leaf area (cm2) Leaf dry weight per cm2 leaf area AT/AC 0.76 ± 0.063b 6.97 ± 0.573bc 217 ± 12c 0.00343 ± 0.00014a AT/EC 1.00 ± 0.071a 11.61 ± 0.814a 322 ± 18a 0.00306 ± 0.00031b ET/AC 0.54 ± 0.034c 6.11 ± 0.400c 196 ± 12c 0.00277 ± 0.00016b ET/EC 0.92 ± 0.041a 8.14 ± 0.411b 263 ± 10b 0.00351 ± 0.00024a (df) F-values, P-values  Temperature (3,76) 7.35, 0.0089 (3,76) 13.95, 0.0004 (3,76) 9.32, 0.0035 (3,76) 0.78, 0.3799  CO2 (3,76) 33.34, <0.0001 (3,76) 33.17, <0.0001 (3,76) 41.88, <0.0001 (3,76) 2.76, 0.1021  Temperature × CO2 (3,76) 1.74, 0.1931 (3,76) 5.10, 0.0278 (3,76) 2.06, 0.1571 (3,76) 24.02, <0.0001 Growth environment Dry weight (g) Fresh weight (g) Leaf area (cm2) Leaf dry weight per cm2 leaf area AT/AC 0.76 ± 0.063b 6.97 ± 0.573bc 217 ± 12c 0.00343 ± 0.00014a AT/EC 1.00 ± 0.071a 11.61 ± 0.814a 322 ± 18a 0.00306 ± 0.00031b ET/AC 0.54 ± 0.034c 6.11 ± 0.400c 196 ± 12c 0.00277 ± 0.00016b ET/EC 0.92 ± 0.041a 8.14 ± 0.411b 263 ± 10b 0.00351 ± 0.00024a (df) F-values, P-values  Temperature (3,76) 7.35, 0.0089 (3,76) 13.95, 0.0004 (3,76) 9.32, 0.0035 (3,76) 0.78, 0.3799  CO2 (3,76) 33.34, <0.0001 (3,76) 33.17, <0.0001 (3,76) 41.88, <0.0001 (3,76) 2.76, 0.1021  Temperature × CO2 (3,76) 1.74, 0.1931 (3,76) 5.10, 0.0278 (3,76) 2.06, 0.1571 (3,76) 24.02, <0.0001 AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Dry weight, fresh weight, and leaf area (means ± SE; N = 20 plants per treatment). Statistical analysis was performed with a two-way ANOVA. Numbers with the same lowercase are not significantly different (P > 0.05). View Large Regarding foliar quality, elevated CO2 resulted in an increase of carbon to nitrogen (C/N) ratio and total nonstructural carbohydrate (TNC); elevated CO2 doubled C/N ratio at ambient temperature and increased by about 30% at elevated temperature. Similarly, elevated CO2 at ambient temperature and elevated temperature increased TNC from 20 to 30% which is 50% increase. On the other hand, elevated CO2 significantly reduced the nitrogen contentat at ambient temperature (0.835%) and elevated temperature (1.094%) (Table 2). Although elevated temperature yielded a slight increase in nitrogen content, it exerted less of an effect on plant nutrition quality compared with elevated CO2. Moreover, temperature and CO2 did not exert significant interactive effects on nitrogen content, TNC and C/N ratio (Table 2). Table 2. Foliar quality of broccoli plants grown under different temperature and CO2 conditions Growth environment Nitrogen content (%) Total nonstructural carbohydrate (%) C/N ratio AT/AC 1.574 ± 0.177b 21.73 ± 0.38c 14.94 ± 1.50b AT/EC 0.835 ± 0.100c 31.22 ± 0.62ab 30.81 ± 1.8a ET/AC 1.978 ± 0.116a 26.57 ± 0.39bc 18.07 ± 1.7b ET/EC 1.094 ± 0.221bc 35.49 ± 1.11a 23.57 ± 0.6ab (df) F-values, P-values  Temperature (3,76) 12.66, 0.0098 (3,76) 2.00, 0.1728 (3,76) 1.30, 0.2724  CO2 (3,76) 26.29, <0.0001 (3,76) 15.66, 0.0008 (3,76) 12.41, 0.0031  Temperature × CO2 (3,76) 0.66, 0.3723 (3,76) 2.44, 0.1338 (3,76) 1.80, 0.1931 Growth environment Nitrogen content (%) Total nonstructural carbohydrate (%) C/N ratio AT/AC 1.574 ± 0.177b 21.73 ± 0.38c 14.94 ± 1.50b AT/EC 0.835 ± 0.100c 31.22 ± 0.62ab 30.81 ± 1.8a ET/AC 1.978 ± 0.116a 26.57 ± 0.39bc 18.07 ± 1.7b ET/EC 1.094 ± 0.221bc 35.49 ± 1.11a 23.57 ± 0.6ab (df) F-values, P-values  Temperature (3,76) 12.66, 0.0098 (3,76) 2.00, 0.1728 (3,76) 1.30, 0.2724  CO2 (3,76) 26.29, <0.0001 (3,76) 15.66, 0.0008 (3,76) 12.41, 0.0031  Temperature × CO2 (3,76) 0.66, 0.3723 (3,76) 2.44, 0.1338 (3,76) 1.80, 0.1931 AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Nitrogen, TNC, C/N ratio (means ± SE; N = 20 plants per treatment). Statistical analysis was performed with a two-way ANOVA. Numbers with the same lowercase are not significantly different (P > 0.05). View Large Table 2. Foliar quality of broccoli plants grown under different temperature and CO2 conditions Growth environment Nitrogen content (%) Total nonstructural carbohydrate (%) C/N ratio AT/AC 1.574 ± 0.177b 21.73 ± 0.38c 14.94 ± 1.50b AT/EC 0.835 ± 0.100c 31.22 ± 0.62ab 30.81 ± 1.8a ET/AC 1.978 ± 0.116a 26.57 ± 0.39bc 18.07 ± 1.7b ET/EC 1.094 ± 0.221bc 35.49 ± 1.11a 23.57 ± 0.6ab (df) F-values, P-values  Temperature (3,76) 12.66, 0.0098 (3,76) 2.00, 0.1728 (3,76) 1.30, 0.2724  CO2 (3,76) 26.29, <0.0001 (3,76) 15.66, 0.0008 (3,76) 12.41, 0.0031  Temperature × CO2 (3,76) 0.66, 0.3723 (3,76) 2.44, 0.1338 (3,76) 1.80, 0.1931 Growth environment Nitrogen content (%) Total nonstructural carbohydrate (%) C/N ratio AT/AC 1.574 ± 0.177b 21.73 ± 0.38c 14.94 ± 1.50b AT/EC 0.835 ± 0.100c 31.22 ± 0.62ab 30.81 ± 1.8a ET/AC 1.978 ± 0.116a 26.57 ± 0.39bc 18.07 ± 1.7b ET/EC 1.094 ± 0.221bc 35.49 ± 1.11a 23.57 ± 0.6ab (df) F-values, P-values  Temperature (3,76) 12.66, 0.0098 (3,76) 2.00, 0.1728 (3,76) 1.30, 0.2724  CO2 (3,76) 26.29, <0.0001 (3,76) 15.66, 0.0008 (3,76) 12.41, 0.0031  Temperature × CO2 (3,76) 0.66, 0.3723 (3,76) 2.44, 0.1338 (3,76) 1.80, 0.1931 AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Nitrogen, TNC, C/N ratio (means ± SE; N = 20 plants per treatment). Statistical analysis was performed with a two-way ANOVA. Numbers with the same lowercase are not significantly different (P > 0.05). View Large Figure 1 shows the development of S. litura from larval stage to pupal stage under different temperature and CO2 conditions. The results demonstrated that S. litura larvae, which fed on broccoli plants grown at elevated temperature and CO2 concentration, had considerably changed development time and growth rate. Elevated CO2 prolonged the insect development, while elevated temperature shortened the insect development, However, combined elevated CO2 and temperature treatment, CO2 did not affect the insect development. Insect growth rate increased when grown under elevated temperature and the growth rate was higher when grown at combining elevated temperature and CO2 treatment. Fig. 1. View largeDownload slide Development of S. litura. Development time from the first instar to pupal stage, pupal weight, and growth rate of S. litura reared and fed on broccoli leaves grown under different temperature and CO2 conditions (N = 15). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Fig. 1. View largeDownload slide Development of S. litura. Development time from the first instar to pupal stage, pupal weight, and growth rate of S. litura reared and fed on broccoli leaves grown under different temperature and CO2 conditions (N = 15). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Larval development was shorter by 5–6 d and the growth rate was significantly increased by 15% at elevated temperature compared with the corresponding measurements under ambient temperature conditions. In addition, elevated CO2 significantly reduced the pupal weight by more than 50 mg, compared with the ambient CO2 treatment. Furthermore, temperature and CO2 had significant interactive effects on the S. litura larval development time and growth rate (Fig. 1). The results of Sn. manilae performance revealed that elevated temperature and CO2 would affect Sn. manilae development. At an elevated temperature, the egg–larval and cocoon development times decreased by 11–12 d and 2–3 d, respectively (Fig. 2). Moreover, elevated temperature significantly increased the cocoon weight and parasitism rate. In addition, elevated CO2 significantly reduced the egg–larval development time by nearly 5 d. Temperature and CO2 exerted significant interactive effects on egg–larval development time (Figs. 2 and 3). Fig. 2. View largeDownload slide Development of Sn. manilae. Egg–larval and cocoon development times and cocoon weight under different temperature and CO2 conditions (N = 30). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Fig. 2. View largeDownload slide Development of Sn. manilae. Egg–larval and cocoon development times and cocoon weight under different temperature and CO2 conditions (N = 30). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Fig. 3. View largeDownload slide Parasitism rate of Sn. manilae under different temperature and CO2 conditions (N = 8). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Fig. 3. View largeDownload slide Parasitism rate of Sn. manilae under different temperature and CO2 conditions (N = 8). AT/AC: ambient temperature/ambient CO2; AT/EC: ambient temperature/elevated CO2; ET/AC: elevated temperature/ambient CO2; ET/EC: elevated temperature/elevated CO2. Bars with the same lowercase are not significantly different (P > 0.05). Discussion The present study demonstrated the effects of elevated temperature and CO2 on a tritrophic system involving broccoli, S. litura and Sn. manilae. Elevated CO2 mainly alters the biomass production and nitrogen content, TNC and C/N ratio of broccoli. The bioassay results reveal that the S. litura fed on elevated CO2-treated broccoli leaves exhibited negative performance with a longer larval development time and decreased pupal weight. The longer larval development times of the host typically related with reduced leaf quality may also extend window time for parasitoids to attack host (Stiling and Cornelissen, 2007). In addition, elevated temperature can directly stimulate the growth and development of S. litura and their parasitoid Sn. manilae likely due to an accelerated metabolism (Kooijman 2014, Akbar et al. 2016). The broccoli plants grown under elevated CO2 (1,000 ppm) conditions exhibited increased aboveground biomass and enlarged leaf area of almost 50% compared with those grown under ambient CO2 conditions. Similarly, a study by Pandey (2017) revealed that a high CO2 concentration can stimulate underground and aboveground biomass in wheat (Triticum aestivum L. var Kundan). However, the results of the present study demonstrate that elevated temperature reduces the growth-stimulating effects of CO2 on broccoli biomass, thereby suggesting that broccoli might be a thermosensitive plant that requires cooler temperatures to achieve optimal growth. Therefore, an increase in temperature of up to 5°C could substantially reduce broccoli biomass. Ciancaleoni (2016) studied broccoli yield response to environmental factors and reported that broccoli yield is largely determined by climate conditions during the growing season and deviations from optimal conditions can seriously affect yield. Nitrogen and carbohydrates are necessary for the growth and development of plant tissue (Uchida 2000). In the present study, the broccoli plants grown under elevated CO2 conditions had a lower total nitrogen content, whereas their TNC concentration (starch and sugar) increased by nearly 10%. Furthermore, our results demonstrate that the C/N ratio was higher in the elevated CO2-treated plants than in the ambient CO2-treated plants. The plant nutrition quality results are in accordance with several climate change projections (Reich et al. 2014, Sardans et al. 2017). Previous studies have reported that elevated CO2 can stimulate photosynthesis, reduce nitrogen concentration, and lead to an increased C/N ratio (Long et al. 2004, Ainsworth and Rogers 2007, De Souza et al. 2008, Leakey et al. 2009). The TNC concentration in plant tissue is considered an indicator of the overall carbon availability status, whereas the caloric value of plants is considered less vital for insect development because carbohydrates can be synthesized from fats by insects (Behmer 2009). However, nitrogen is essential for insect development, and thus foliage with decreased nitrogen concentration might be considered a poor food source for insects (Caulfield and Bunce 1994). Nevertheless, our results demonstrate that elevated temperature has less of an effect on plant nutrition quality under elevated CO2 conditions. The results of the present study clearly demonstrate that CO2 concentration is a crucial factor influencing herbivore and parasitoid performance. The S. litura fed on elevated CO2-treated broccoli leaves exhibited prolonged larval development time and decreased pupal weight, thereby showing consistency with the findings of previous studies, which have demonstrated that elevated CO2 exerts an indirect effect on insect growth and development by reducing the nutrition quality of the host plant (Srinivasa Rao et al. 2012). Interestingly, our study found that elevated CO2 prolonged larval development time of S. litura, in contrast elevated CO2 shortened larval development time of Sn. manilae. One possible explanation may be that the elevated CO2 can either increase or decrease production of volatile emissions and plant defensive compounds that can affect the parasitoid development (Heil 2008). For example, the study by Klaiber (2013) indicated that Brassica plants grown under elevated CO2 contained higher levels of glucosinolates which could reduce the body mass of aphids and shorten the parasitoid development time. In addition, one previous study suggested that the improved performance of the parasitoids Spilichneumon limnophilus (Thomson) (Hymenoptera: Ichneumonidae) and Chasmias paludator (Desvignes) (Hymenoptera: Ichneumonidae) is strongly correlated with body size, which is associated with plant vigor (Teder and Tammaru 2002). Our results reveal that although elevated temperature (29/26°C, day/night) has less of an effect on plant nutrition quality than does elevated CO2, it strongly affects insect development. Elevated temperature can increase the larval development time of S. litura. These findings are consistent with those of previous studies. One study reported that elevated temperature hinders S. litura development (Rao et al. 2014). The performance of the parasitoid Sn. manilae and its host S. litura show similar trends, both exhibiting decreased larval and cocoon development times. Moreover, at elevated temperature, the female parasitoids became more physically active, resulting in an increase (<15%) in the parasitism rate. The increased development times of the herbivorous host and increased parasitoid activity at elevated temperature in our study could be explained by Neven (2000), who reported that insect body temperature increases at high temperature with concomitant increases in insect metabolism and respiration, leading to changes in insect development and behaviors. Similarly, previous studies have revealed that Aenasius arizonensis (Girault) (Hymenoptera: Encyrtidae), an endoparasitoid of Phenacoccus solenopsis (Tinsley) (Hemiptera: Pseudococcidae), has the highest parasitism and emergence rates at 31°C (He et al. 2017). In addition, Sn. manilae has improved performance at elevated temperature, which consequently increases the cocoon weight and parasitism rate. These findings are consistent with results of numerous studies that have suggested parasitoids can develop under various thermal conditions because of their mating and host-locating behaviors (Stireman et al. 2005, Thomson et al. 2010). Parasitoids are more likely to encounter variable climate conditions and might have higher potential for adaptability than their herbivorous hosts, particularly in subtropical regions with average temperatures of 20–35°C. In conclusion, elevated temperature and CO2 significantly influenced tritrophic systems possibly by altering host plant nutrient availability, which further affects the growth and development of S. litura and Sn. manilae. We suggest that the dynamic and communal structure of S. litura and its parasitoids must be comprehensively evaluated in terms of the changes in their nutrient quality (bottom-up controlled) caused by variable CO2 and temperature conditions. Our study results could facilitate understanding of the interactions of trophic levels in future environments, particularly in subtropical regions. Acknowledgments The study was supported by grant from Ministry of Science and Technology (MOST), Taiwan (105-2313-B-005-003-MY3). We thank Wallace Academic Editing, Sharon Bailey and Mark Darvill. References Cited Ainsworth , E. A. , and A. Rogers . 2007 . The response of photosynthesis and stomatal conductance to rising [CO2]: mechanisms and environmental interactions . Plant. 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For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

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Environmental EntomologyOxford University Press

Published: Aug 1, 2018

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