Influence of elevated CO2 on development and food utilization of armyworm Mythimna separata fed on transgenic Bt maize infected by nitrogen-fixing bacteria

Background Bt crops will face a new ecological risk of reduced effectiveness against target-insect pests owing to the general decrease in exogenous-toxin content in Bt crops grown under elevated carbon dioxide (CO2). The method chosen to deal with this issue may affect the sustainability of transgenic crops as an effective pest management tool, especially under future atmospheric CO2 level raising. Methods In this study, rhizobacterias, as being one potential biological regulator to enhance nitrogen utilization efficiency of crops, was selected and the effects of Bt maize (Line IE09S034 with Cry1Ie vs. its parental line of non-Bt maize Xianyu 335) infected by Azospirillum brasilense (AB) and Azotobacter chroococcum (AC) on the development and food utilization of the target Mythimna separate under ambient and double-ambient CO2 in open-top chambers from 2016 to 2017. Results The results indicated that rhizobacteria infection significantly increased the larval life-span, pupal duration, relative consumption rate and approximate digestibility of M. separata, and significantly decreased the pupation rate, pupal weight, adult longevity, fecundity, relative growth rate, efficiency of conversion of digested food and efficiency of conversion of ingested food of M. separata fed on Bt maize, while here were opposite trends in development and food utilization of M. separata fed on non-Bt maize infected with AB and AC compared with the control buffer in 2016 and 2017 regardless of CO2 level. Discussion Simultaneously, elevated CO2 and Bt maize both had negative influence on the development and food utilization of M. separata. Presumably, CO2 concentration arising in future significantly can increase their intake of food and harm to maize crop; however, Bt maize infected with rhizobacterias can reduce the field hazards from M. separata and the application of rhizobacteria infection can enhance the resistance of Bt maize against target lepidoptera pests especially under elevated CO2.


INTRODUCTION
With increased fossil fuel combustion and drastic changes in land utilization, the concentration of atmospheric carbon dioxide (CO 2 ) has increased by more than 40%, (Yamprai, Mala & Sinma, 2014). Thus, optimization of soil-nitrogen management offers significant potential in the utilization of soil rhizobacterias to increase Bt-crop nitrogen utilization to affect the expression of Bt toxin under elevated CO 2 .

Setup of CO 2 levels
A two-year study (2016)(2017) was conducted in six open-top chambers (i.e., OTCs; Granted Patent: ZL201120042889.1; 2.5 m in height Â 3.2 m in diameter) (Chen et al., 2011) at the Innovation Research Platforms for Climate Change, Biodiversity and Pest Management (CCBPM; http://www.ccbpm.org) field laboratory in Ningjin County, Shandong Province of China (37 38′ 30.7″ N,116 51′ 11.0″ E). A total of two CO 2 levels, ambient (375 ml/L, hereafter referred to as aCO 2 ) and elevated (750 ml/L or doubleambient, hereafter referred to as eCO 2 ) were applied continuously from 10 June to 7 October in both years. A total of three OTCs were used for each CO 2 treatment, and the CO 2 concentrations in each OTC were monitored continuously and adjusted using an infrared CO 2 analyzer (Ventostat 8102; Telaire Company, Goleta, CA, USA). The OTCs of elevated CO 2 treatments were inflated with canned CO 2 gas with 95% purity and automatically controlled by the same type of infrared CO 2 analyzer (Chen, Wu & Ge, 2004). Actual mean CO 2 concentrations and temperature throughout the entire experiment for both 2016 and 2017 are provided in Table 1.

Plant materials
The Bt maize cultivar (Line IE09S034, hereafter referred to as Bt) and its non-Bt parental line (cv. Xianyu 335, referred to as Xy) were both obtained from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Both Bt and non-Bt lines used in this study had the similar maturity (approximately 102 d: from 10 June to 20 September) and were well adapted to the growing conditions of northern China (Guo et al., 2016;Zhang et al., 2013;Jia et al., 2016;Ling, 2010). Both maize accessions were planted in plastic buckets (diameter Â height = 30 Â 45 cm) filled with 20 kg autoclaved soil and OTC: ambient-CO 2 OTC (aCO 2 OTC) and elevated-CO 2 OTC (eCO 2 OTC). Different lowercase letters indicate significantly different between the eCO 2 OTC and aCO 2 OTC in same year by the Duncan test at P < 0.05, respectively. A Not significantly different between 2016 and 2017 at same CO 2 level or temperature by the Duncan test at P > 0.05, respectively.

Soil nitrogen-fixing bacteria and infection of maize seeds
Lyophilized Azospirillum brasilense (strain number ACCC 10103) and Azotobacter chroococcum (strain number ACCC 10006) were provided by Agriculture Culture Collection of China (ACCC) in plastic tubes (3 cm in diameter and 15 cm in height) with bacterial growth medium. Both species of rhizobacterias were grown in liquid medium at 28 C under continuous shaking (200 rpm) until they reached an absorbance of 1.008 (A. brasilense) and 1.005 (A. chroococcum) at a wavelength of 600 nm. Before inoculation, the culture was centrifuged, and the supernatant was discarded, and the pellet of cells was re-suspended in the liquid medium to a density of 10 8 copies per milliliter. The seeds of both Bt and non-Bt maize were infected with A. brasilense and A. chroococcum cultures each, and the inoculation doses were all adjusted to a final volume of 10 ml for each seed. After inoculation, all the treated seeds were maintained under sterile laminar air flow for 2 h at 28 C (Cassán et al., 2009). Bacteria inoculation treatments consisted of three types of rhizobacteria infection, including (1) seeds infected with A. brasilense (referred to as AB); (2) seeds infected with A. chroococcum (referred to as AC); and (3) non-infected seeds (control) treated with a final volume of buffer solution (referred to as CK). The entire experiment, thus, consisted of 12 treatments, including two CO 2 levels (aCO 2 and eCO 2 ), two maize cultivars (Bt and Xy), and three rhizobacteria infections (AB, AC, and CK), replicate six time. Each pot serves as one replication. Specifically, six buckets for each maize cultivar (Bt and Xy) and three rhizobacteria inoculations (6 buckets per transgenic treatment Â 2 transgenic treatments Â 3 inoculation treatments = 36 buckets) were placed randomly in each CO 2 chamber (ambient and double-ambient CO 2 ), and three maize seeds were sown in each bucket at 2 cm soil depth. No pesticides were applied during the entire experimental period and the manual weeding keep the maize buckets weed-free during the experiment. The rhizosphere soil was sampled from each bucket one-day before planting, 14 days after planting, and at harvest and measured the relative density of A. brasilense and A. chroococcum using RT-PCR (Tables 2 and 3) (Jiang et al., 2017).

Note:
Rhizobacteria infections: A. brasilense (AB) and A. chroococcum (AC) vs. the control buffer solution (CK). CO 2 levels: ambient CO 2 (aCO 2 ) and elevated CO 2 (eCO 2 ). Transgenic treatment: Bt maize (Bt) and non-Bt maize (Xy). Different lowercase letters indicate significantly different between ambient CO 2 and elevated CO 2 for same maize cultivar in same year by the Duncan test at P < 0.05, respectively. size for the M. separata larval feeding trial consisted of 20 larvae (sample unit size) with five replicates for each of the 12 treatment combinations (i.e., 1,200 larvae evaluated for the entire study). Because of the cannibalism among the late instar larvae of M. separata (Jiang et al., 2016;Ali et al., 2016;Liu et al., 2017), the sampled larvae were reared separately in the Petri dish until pupation.

Development and reproduction of M. separata
Larval development was evaluated from third instar to pupation by way of observing each individual petri dish every 8 h and recording the timing of larval ecdysis, pupation, and emergence of M. separata moths. After eclosion, the newly emerged moths were paired (female: male = 1:1) for mating in a metal frame screen cage (length Â width Â height = 35 Â 35 Â 40 cm), and the paired moths were fed with a 10% honey solution provided on a large cotton wick in a single plastic cup (diameter Â height = 8 Â 20 cm) covered with cotton net yarn butter paper for oviposition. The cotton net yarn and butter paper were replaced every day. Moth survivorship and oviposition were recorded daily until both moths from each pair died.

Food utilization of the larvae of M. separata
Each third instar test larvae of M. separate was weighed at the initiation of the feeding trial by using an electronic balance (AL104; METTLER-TOLEDO, Greifensee, Switzerland). Total accumulated feces from third instar until the larva entered pupal stage (sixth instar), sixth instar larval weight, and the remaining leaves were also weighed. The food utilization indices of M. separata included the relative growth rate (RGR), relative consumption rate (RCR), approximate digestibility (AD), efficiency of conversion of ingested food (ECI) and efficiency of conversion of digested food (ECD) (Chen, Ge & Parajulee, 2005a;Chen et al., 2005b). Formulas for calculation of the measured indices were adapted from Chen et al. (2005b).

Data analysis
All data were analyzed using the statistical software SPSS 19.0 (2015; SPSS Institute, Chicago, IL, USA). Four-way analysis of variance was used to analyze the effects of CO 2 levels (elevated vs. ambient), transgenic treatment (Bt maize vs. non-Bt maize), rhizobacteria infection (AB and AC vs. CK), sampling years (2016 vs. 2017), and the interactions on the measured indices of growth, development, and reproduction, including larval life-span, pupation rate, pupal weight, pupal duration, adult longevity and fecundity of M. separata. The measured food utilization indices were analyzed by using an analysis of covariance with initial weight of M. separata (i.e., third instar larva) as a covariate for RCR and RGR, while food consumption was a covariate for ECI and AD to correct the effect of variation in the growth and food assimilation of M. separata (Raubenheimer & Simpson, 1992); food assimilated was also used as a covariate to analyze the ECD parameter (Hägele & Rowell-Rahier, 1999). The assumption of a parallel slope between covariate and dependent variable was satisfied for each analysis. Treatment means were separated by using the Duncan-test to examine significant difference at P < 0.05.

RESULTS
Effects of CO 2 level, transgenic treatment, and rhizobacteria infection on the rhizosphere soil densities of A. brasilense and A. chroococcum in different sampling period Significant effects of rhizobacteria infection (P < 0.001) were observed on the measured rhizosphere soil densities of A. brasilense (AB) and A. chroococcum (AC) 14 days after maize planting. Compared with ambient CO 2 , elevated CO 2 significantly increased the rhizosphere soil densities of both A. brasilense and A. chroococcum; compared with the control buffer solution (CK), rhizobacteria infection significantly increased the rhizosphere soil densities of A. brasilense and A. chroococcum (P < 0.001; Table 3). CO 2 level and rhizobacteria infection both significantly affected the densities of A. brasilense and A. chroococcum in rhizosphere soil at maize harvest (Table 4).
Impacts of CO 2 level, transgenic treatment, and rhizobacteria infection on the food utilization of M. separata There were significant effects of CO 2 level, transgenic treatment, and rhizobacteria infection (P < 0.01 or P < 0.001) on food utilization of M. separata fed on both Bt and non-Bt maize infected with A. brasilense and A. chroococcum at both CO 2 levels in both years of the study (Table 7).  Tables 4 and 5. e Initial weight as a covariate for RGR and RCR, and food consumption as a covariate for AD and ECI, and food assimilated as a covariate for ECD.
Interactive influence of CO 2 level, transgenic treatment, and rhizobacteria infection on growth, development and reproduction of M. separata In addition to the significant main effects of CO 2 level, transgenic treatment, and rhizobacteria infection, there were significant two-way and three-way interaction of these three main effects on larval life-span, pupation rate, pupal weight and duration, adult longevity, and fecundity of M. separata fed on Bt and non-Bt maize infected with A. brasilense and A. chroococcum under both CO 2 levels in both years of the study (P < 0.05, P < 0.01 or P < 0.001; Table 5).

Transgenic treatment Â CO 2 Â Rhizobacteria
There were opposite trends in the measured food utilization indexes of M. separata larvae fed on Bt maize (Bt) and non-Bt maize infected with A. brasilense (AB) and A. chroococcum (AC) compared with the CK in both years regardless of CO 2 level (Fig. 4).
In comparison with the CK, rhizobacteria infection with A. brasilense and A. chroococcum both significantly decreased RGR, ECD, and ECI of M. separata fed on Bt maize, and significantly increased RGR, ECD, and ECI of M. separata fed on non-Bt maize under the same CO 2 level; and rhizobacteria infection with A. brasilense and A. chroococcum both significantly enhanced RCR and AD of M. separata fed on Bt maize, and significantly reduced RCR and AD of M. separata larvae fed on non-Bt maize under the same CO 2 level. Moreover, compared with ambient CO 2 , elevated CO 2 significantly increased RCR and AD, and significantly decreased RGR, ECD, and ECI of M. separata larvae fed on same type of maize cultivar infected with A. brasilense and A. chroococcum in both years (P < 0.05; Fig. 4). Furthermore, there were significant decreases in RGR, ECD, and ECI, and significant increases in RCR and AD of M. separata larvae fed on Bt maize in contrast to non-Bt maize infected with same type of rhizobacteria species within the same CO 2 level in both years.

DISCUSSION
Insects are sensitive to environmental variations, and environmental stresses can cause changes on their growth, development, fecundity, food utilization and the occurrence and distribution of populations as a result of metabolic rate fluctuation (Bloom et al., 2010). In this study, elevated CO 2 significantly prolonged larval and pupal duration and decreased pupation rate and pupal weight of M. separata compared to ambient CO 2 . Elevated CO 2 negatively affected the larval survival, weight, duration, pupation, and adult emergence of cotton bollworm, H. armigera (Akbar et al., 2016), and reduced the egg laying by Cactus moth Cactoblastis cactorum (Stange, 1997) and Achaea Janata (Rao et al., 2013). In this study, elevated CO 2 significantly increased the RCR (+10.44%) and the AD (+5.59%) (i.e., AD), and significantly reduced the RGR (-9.95%), ECD (-16.05%) and ECI (-17.95%) of M. separata larvae compared with ambient CO 2 . RGRs of Gypsy moth (Lymantria dispar) were reported to be reduced by 30% in larvae fed on Quercus petraea exposed to elevated CO 2 (Hattenschwiler & Schafellner, 2004). RCR was significantly higher for H. armigera larva fed maize grown at 375 and 750 ppm CO 2 in contrast to ambient CO 2 condition, and elevated CO 2 significantly decreased the ECI food, the ECD food, and the RGR of H. armigera larvae compared with ambient CO 2 (Yin et al., 2010). According to the "Nutrition compensation hypothesis," elevated CO 2 can affect the development fitness of herbivores by changing the nutritional components, above and below-ground biomass, and photosynthetic rate of host plants indirectly Jackson et al., 2009;Zavala, Nabity & Delucia, 2013), including increased C/N ratio and decreased nitrogen content etc. Declined growth rate, reproduction, and survival rate were found in the chewing mouthparts insects (e.g., H. armigera, Spodoptera exigua, M. separata), and the food consumption of which increased so that they could obtain necessary nutrition to survive (Bottomley, Rogers & Prior, 1993;Rogers et al., 2006). Yin et al. (2010) reported that elevated CO 2 increased the food consumption and prolonged the development time of H. armigera, which due to the reduced nutritional quality of maize leaves, as a result of reduced nitrogen content and increased C/N ratio. Elevated CO 2 significantly reduced the food conversion rate and enhanced the food ingestion of H. armigera, which attribute to reduced nitrogen content of the cotton, Simian-3 (Chen, Ge & Parajulee, 2005a;Chen et al., 2005b). Thus, Chen, Ge & Parajulee (2005a) and Chen et al. (2005b) inferred that elevated CO 2 might be unfavorable to H. armigera. Our results in maize system appear to be similar to the study by Chen, Ge & Parajulee (2005a) and Chen et al. (2005b) in a cotton system. Although the transgenic corn, Zea mays L., hybrids expressing the Cry insecticidal protein from Bacillus thuringiensis (Bt) were developed to control H. zea, O. nubilalis, S. frugiperda, and M. separata (Koziel et al., 1993;Armstrong et al., 1995;Jouanin et al., 1998;Lynch, Plaisted & Warnick, 1999), few studies focused on the defense responses of transgenic cry1Ie maize to corn armyworm under elevated CO 2 , especially on the growth, development and food utilization of the pest insects. Prutz & Dettner (2005) reported that the transgenic Bacillus thuringiensis-maize could result in decreased growth rate and increased mortality, which might attribute to the termination of larval metamorphosis. Most studies showed that adverse effects on life-table parameters of different herbivores were direct by the Cry protein (Lawo, Wäckers & Romeis, 2010), which might be due to the interaction of feeding inhibitors and growth inhibitors (e.g., secondary plant substances) (Smith & Fischer, 1983). Effects of elevated CO 2 on the plant nutrition, metabolism and secondary defense metabolism might adverse for the growth, development and nutrition utilization of herbivores (Akbar et al., 2016). The insects possessed more nutrients to meet their growth needs and prolong the food digestion time in the midgut so that the RCR and AD increased (Reynolds, Nottingham & Stephens, 1985). In this study, we found that some negative effects of transgenic cry1Ie maize (Bt) and Xianyu 335 (Xy) grown in elevated CO 2 on the food utilization indices (including RGR, ECD, and ECI) of M. separata larvae and some positive effects on the RCR and AD, which indicated that the resistance responses of Bt maize might persist under elevated CO 2 , and M. separata might ingest more food to get enough nutrition for surviving in limited developmental time under elevated CO 2 . Meanwhile the Bt maize and its parental line (Xianyu 335) prolonged their larval life-span and pupal duration, decreased growth rate and increased mortality that might result in lowering of pests' occurrence. According to the "carbon nutrition balance hypothesis" (Gebauer, Strain & Reynolds, 1997), elevated CO 2 would increase the fixed organic matter in plant while increase C-based secondary metabolites and decrease N-based secondary metabolites, thus affecting the insects resistance of plants. Robinson, Ryan & Newman (2012) indicated that elevated CO 2 increased 19% phenols, 22% condensed tannins, and 27% flavonoids, while the terpenoids and NBSC decreased by 13% and 16% respectively. Coviella, Stipanovic & Trumble (2002) anticipated that the primary CO 2 effect on Bt toxin production would be due to differences in N concentration within the plant. In a meta-analytical review of 33 studies that simultaneously increased CO 2 conditions compared to ambient conditions, Zvereva & Kozlov (2006) showed that nitrogen concentration in plants was reduced under elevated CO 2 , and this decrease was stronger for woody compared to herbaceous plants.
If conditions of increased carbon (e.g., elevated CO 2 ) allow plants to allocate significantly more resources to condensed tannins and gossypol, then the enzyme composition in the insect herbivore is expected to also change. Similarly, if Bt toxin production changes due to elevated CO 2 , then the insect herbivore's body enzymes should also be changed in this circumstance.
Most of the nitrogen, however, is found in the form of N 2 which approximately amounts to 78% in the atmosphere. As plants cannot use this form of nitrogen directly, some microbes can change the N 2 into ammonia. Most free living microbes in soil which can fix nitrogen and whose activities in enhancing the growth of plants are bacteria namely Azotobacter sp. and Azospirillum sp. These two bacteria are particularly important in maize production system due to their greater nitrogen fixing ability. Azospirillum acquires carbohydrate directly from sieve tube as a resource of carbon which promotes its growth (Olivera et al., 2004). Azospirillum can be used to promote the growth of sprouts under normal and arid conditions (Alejandra et al., 2009). Azospirillum also provides more flexibility to cell wall which enhances the growth (Pereyra et al., 2010) and increases products of wheat in waterless plot of land (Martin, 2009). Furthermore, azospirillum had the highest efficiency in nitrogen fixation at the root of sweet corn and it would reach the highest point of nitrogen fixation in the week 4 amounting to 0.20 mgNhr -1 m -2 (Toopakuntho, 2010). Azospirillum can also create auxin, a substance promoting growth of maize, of 53.57 mg/ml (Phookkasem, 2011). Therefore, we used techniques of rhizobacteria (A. brasilense and A. chroococcum) inoculation of maize seeds to stimulate plant N uptake to increase in biomass N relative to C under elevated CO 2 , increase Bt toxin production for transgenic cry1Ie maize and create a substance promoting maize plant growth. In this study, we found that elevated CO 2 significantly enhanced the rhizosphere soil densities both A. brasilense and A. chroococcum at the maize harvest, but there was no significant difference of the rhizosphere soil densities both A. brasilense and A. chroococcum between elevated and ambient CO 2 at the maize seedling after 14 days. We hypothesize that the elevated CO 2 increased the maize root bifurcation and soil nutrition (e.g., carbohydrates, amino acids and multi-trace elements) for rhizobacteria to provide the living space and nutrition with a long-time environmental effect. Other researchers have also shown positive effects of elevated CO 2 on the bacterial community in the rhizosphere of maize (Chen et al., 2012). Moreover, significant adverse effects on the growth, development, reproduction, and food utilization of M. separata were observed when the host substrate maize was exposed to rhizobacteria treatments, which might be attributed to rhizobacteria stimulating plant N uptake to increase Bt toxin production for transgenic cry1Ie maize and promoting growth of its parental line (Xianyu 335) (Olivera et al., 2004;Stitt & Krapp, 1999).
There was no significant year-to-year variation in our field research data. Therefore, the overall results clearly indicate that increasing CO 2 had negative effects on M. separata. Resistance performance of transgenic cry1Ie maize decreased under elevated CO 2 as shown by decreased RGR, ECD, and ECI. The rhizobacteria treatments (A. brasilense and A. chroococcum) had positive effects on improving the effectiveness of Bt maize on target Lepidoptera pest management via decreased RGR, ECD, and ECI of M. separata that fed on transgenic cry1Ie maize and promoting growth of Xianyu 335 via increased RGR, ECD, and ECI of M. separata. Under future predicted climate changes (e.g., elevated CO 2 ), it is particularly important to understand the field insect resistance traits of resistant crops to target pests. In an environment of accelerated greenhouse effect, Bt maize may have decreased resistance performance in the field with inhibiting effect on the development and food utilization of insects. Therefore, we used techniques of rhizobacteria (A. brasilense & A. chroococcum) inoculation of maize seeds to stimulate plant N uptake to increase in biomass N relative to C under elevated CO 2 , increase Bt toxin production for transgenic cry1Ie maize, and create a substance promoting maize growth.

CONCLUSION
Overall, our results indicated that elevated CO 2 and Bt maize were negative against development and food utilization of M. separata. Rhizobacteria infection significantly increased the larval life-span, pupal duration, RCR and AD of M. separata, and significantly decreased RGR, ECD and ECI of M. separata fed on Bt maize; there were opposite trends in development and food utilization of M. separata fed on non-Bt maize infected with rhizobacterias compared with the CK in 2016 and 2017 regardless of CO 2 level. This study demonstrates that the use of rhizobacteria (e.g., A. brasilense and A. chroococcum) as pest control enhancer especially under elevated CO 2 is significantly more beneficial in transgenic Bt maize system compared to that in non-transgenic system. Rhizobacteria (A. brasilense & A. chroococcum), as being one potential biological regulator to enhance nitrogen utilization efficiency of crops, could make the Bt maize facing lower field hazards from the target pest of M. separate, and finally improve the sustainability and resistance of Bt maize against target lepidoptera pests, especially under future CO 2 raising.