Evaluation of the Ecological Safety of Polygenic Cotransformed Populus × euramericana

Evaluation of the Ecological Safety of Polygenic Cotransformed Populus × euramericana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of the Ecological Safety of Polygenic Cotransformed Populus × euramericana Yali Huang, Zijie Zhang, ChengCheng Li, Shijie Wang, WeiZhen Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6954149/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted 11 You are reading this latest preprint version Abstract BACKGROUND: Poplar (Populus SPP.) is an important model species in forest research. With the development of transgenic technology, the genetic transformation of poplar with multi-gene resistance to insect pests and saline-alkali stress has been successfully realized. Here, test-forests were established in Baoding City, Cangzhou City, and Tangshan City of Hebei Province to compare the arthropod community of wildtype and transgenic polygene 107 poplar (GM 107) in the field, and thus determine whether the performance of transgenic poplar with field pest resistance and saline-alkali tolerance is ecologically safe. RESULTS: The results confirmed the presence of exogenous genes of field-transgenic poplar GM 107 and the stable expression of the corresponding toxic proteins. The arthropod communities of GM 107 varied among different regions in the same year and among different years in the same region. Additionally, GM 107 showed high insect resistance and selectivity. Toxic effects on Lepidoptera were as follows: Micromelalopha troglodyta > Hyphantria cunea > Clostera anachoreta > Lymantria dispar. The toxic effects on Coleoptera were as follows: Plagiodera versicolora > Cerambycidae. Additionally, there was no obvious inhibitory or proliferative effect on natural enemies or neutral insect populations. Arthropods of field-transgenic poplar varied greatly among regions and vintages. Further, upon a severe lepidopteran pest outbreak, the arthropod community of transgenic ecosystems were more stable, and the structures were similar compared to CK. CONCLUSION: GM 107 effectively showed an increased ability of the arthropod community. Our study provides strong theoretical support for the safe and sustainable application of GM 107. Transgenic Populus × euramericana Bacillus thuringiensis genes Ecological safety Arthropod Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Owing to its fast growth and wide range of uses, poplar is currently being extensively planted worldwide. Nonetheless, as a consequence of the extensive use of only a few poplar clones, a decrease in poplar genetic diversity and serious insect pest-infestation problems have been reported in recent years (Wang et al., 2018). Additionally, owing to the large area with saline-alkali soils in China and the shortage of saline-alkali resistant germplasm, breeding for poplar resistance is an increasingly urgent need. However, the long reproductive cycle and complex reproductive characteristics of this species greatly limit successful breeding of new poplar varieties. Beginning in the 20th century, genetic engineering-assisted plant breeding, has proved effective in accomplishing great directional variety improvement in a short time. Furthermore, it is an effective way to solve the problems of poplar insect infestation and restrictions to a wider geographical distribution (Noushahi and Hussain, 2020). Some elite poplar-hybrid clones have been successively cultivated in China and have entered the stages of intermediate testing, environmental release, production testing, and commercial application (Hu et al., 2010). Insect pest resistance adaptability and non-target insect toxicity in different geographical and climatic environments are key factors for assessing the ecological impact of Bt transgenic plants (Tabashnik et al., 2013; Martinez et al., 2018). Indeed, to date, many countries have already conducted large-scale environmental release tests on Bt transgenic plants, and the first lepidopteran pest resistance to Bt preparation in the world was reported in 1985. Subsequently, a number of laboratory and field studies have confirmed that a range of insects, including Lepidoptera, Coleoptera, Diptera, and nematode species, have developed resistance to the Bt toxin under long-term high-pressure selection (Marroquin and Elyassnia, 2000; Ferré and Van Rie, 2002; Tabashnik and Carrière, 2017; Chandrasena et al., 2018). Tabashnik et al. (1990) found resistance in diamondback moths to Bt preparations in field tests conducted in Hawaii, USA, where Bt preparations have been used for a long time. Additionally, after monitoring the susceptibility of Spodoptera frugiperda to Cry1F -modified maize in several regions of Argentina for three years, Chandrasena et al. (2018) were the first to report the detection of S. frugiperda resistance to the Cry1F toxin in Argentina, and similar results have been obtained in Brazil (Juliano et al., 2014). Further, a study conducted in 2018 in Europe found that field populations of Sesamia nonagrioides increased the frequency of the Cry1Ab resistance allele to 0.0036 after 12 years of cultivation of Cry1Ab-transgenic corn (Camargo et al., 2018). Serine protease inhibitors (serpins) inhibit the conversion of the Cry1Ac proprotein into the activated toxin, resulting in reduced insecticide activity of Cry1Ac against cotton bollworms (Zhang et al., 2021). In turn, the first forced recall of GM varieties owing to resistance issues was reported in Puerto Rico in 2012 (Storer et al., 2012). Furthermore, Fu et al. (2021) determined the virulence of four insecticidal proteins, Cry1Ab, Cry1Ac, Cry1Ah, and Cry1Ca, against the Btk resistant lines F80 and F100 of cabbage moth and identified the development rules of resistance of cabbage moth- Btk resistant lines to four Bt insecticidal crystal proteins. The resistance of Btk resistant lines to Cry1Ac developed rapidly, followed by that to Cry1Ab, whereas resistance to Cry1Ca and Cry1Ah developed more slowly. Further, Fed leaves of transgenic poplar trees carrying the Cry3A gene to plant-eating leaf beetles under greenhouse conditions. Their experiments showed plant resistance to leaf beetles at all stages. (Liliana et al., 2012). Similarly, Yang et al. (2005) conducted a 4-year indoor insect-feeding experiment using 741 poplar trees with double Bt gene transfer and found that the insect resistance effect of the three high-resistance lines was stable, and the larval mortality rate of lepidoptera leaf-eating pests such as Clostera anachoreta, Lymantria dispar, Hyphantria cunea was higher than 80%, while the development and cocoon formation of surviving larvae were strongly hampered. Consistently, Wang et al. (2014) fed H. cunea with the leaves of 1-year old poplar seedlings carrying the Bt gene. Although the mortality rates of the different transgenic clones differed, they all showed high insecticidal effects. Additionally, Meilan et al. (2000) conducted field trials on 51 Cry3A transgenic poplar trees and found that almost all transgenic lines in eastern Washington showed low insect feeding damage under natural conditions in the wild, whereas non-transgenic lines showed severe leaf damage and an average growth rate that was 13% lower than that of transgenic plants. Furthermore, Chen et al. (2012) investigated the arthropod community of the poplar and cotton complex ecosystem and found that, compared with other complex ecosystems, the transgenic poplar and cotton complex ecosystem had a strong compound inhibitory-effect on the target pests. Altogether, the above studies indicate that the ecological effects of Bt transgenic plants on the ecosystem in the field cannot be generalized. In the early stages, we linked insect-resistant and saline-alkali-resistant genes into the same vector and introduced them into the poplar genome using the Agrobacterium -mediated transformation method, which successfully achieved a breakthrough in the breeding of insect-resistant and saline-alkali-resistant poplar cultivars (Liu et al., 2016; Yang et al., 2016). Moreover, transgenic poplars performed well in insect-feeding experiments. In this study, the arthropod community of transgenic polygene 107 poplar (GM 107) was analyzed at different times and in different spaces through a regionalization experiment. The rationale of our study is that it is of the greatest theoretical and practical significance to further study the effects of exogenous gene introduction on the arthropod community, explore the ecological relationship between plants and insects, and control harmful insect populations. 2 Methodology 2.1 Research material The transgenic polygene Populus × euramericana (referred to as GM 107) was cultivated at Hebei Agricultural University. Three lines carrying Cry1Ac-Cry3A-BADH genes (referred to as A1-A3), three lines with Cry1Ac-Cry3A-NTHK1 genes (referred to as B1-B3), and the receptor 107 poplar were used as research materials (Liu et al., 2016; Yang et al., 2016). The vector maps of the C ry1Ac-Cry3A-BADH and Cry1Ac-Cry3A-NTHK1 genes are shown in Supplementary Figure 1. All six transgenic lines were approved by the National Forestry and Grassland Administration for environmental release planting, and the high-resistance lines were screened as A1 and B1 in the indoor feeding experiments. Experiment design In April 2018, experimental forests of GM 107 were established in Baoding City, Cangzhou City, and Tangshan City in Hebei Province (Figure 1). A completely randomized blocks design was adopted, and non-GM 107 plants were planted around each block as a border line. The terrain, geomorphology, soil quality, air temperature, vegetation, and cultivation management conditions in the blocks were consistent. 2.2 Measuring insect resistance Insect resistance was determined at 30-day intervals during the growing season. Six lines were investigated using a random sampling method and five plants were randomly selected from each block of each line. In all east, south, west, and north directions, we recorded the species and number of arthropods in the upper and lower branches, on the trunk within 2 m of the soil surface, and a 1 m×1 m ground surface area around the trunk. For mites and aphids present in large numbers, the number of insects on the third leaf from the tip of the sampled branch was recorded. Most insects were identified to the species level but some only to the family level. According to their feeding characteristics, arthropods in the community were divided into basal, median, top, and into three-nutrient levels (Blondel, 2003). Based on their systematic classification, spatial distribution, and feeding habits, insects in the arthropod communities were divided into different functional groups. 2.3 Community feature index Total number of individuals ( N ): the sum of the individuals counted in each tree line. Richness ( S ): The total number of arthropod species in the community. Relative abundance: P i = N i /N , where N i is the number of individuals of the ith species and N is the total number of individuals. Diversity Index ( H' ): Shannon-Wiener Diversity index is adopted and the formula is: Community evenness € was calculated using the evenness index proposed by Pielou as follows: Berger-Parker dominance index: C = N max / N where, N max is the number of dominant species; N is the number of individuals of all species. 2.4 Community stability The ratios of total community species to total community individuals ( S s /S t ), predatory natural enemies to herbivorous insects ( S n /S p ), and predatory natural enemies to herbivorous insects ( S a /S b ) were used to describe the relative stability of the community. While S s /S t reflects the quantitative restrictions among species, S n /S p reflects the complexity of food web relationships within communities. 2.5 Similarity analysis The total number of arthropods of different orders on different transgenic lines during the whole year was considered as a sample, and the differences in arthropod communities in different groups were tested by non-metric multidimensional scaling (NMDS) based on the Bray-Curtis distance coefficient. After standardization of the original data, the European distance and class average method (WPGMA) were used for clustering. 2.6 Primary response curve analysis The principal response curve (PRC) analysis, is a multivariate analysis of the long-term effects of a treatment. It is suitable for the screening of indicator species, and the statistical results are evaluated by Monte Carlo arrangement tests (Van Wijngaarden et al., 1995). 2.7 Statistical analysis Excel was used for statistical analysis of the data. Five surveys were conducted at similar points for inter-year comparative analysis, SPSS was used for variance analysis, R language was used for PRC analysis, and GraphPad Prism software was used for data visualization. For community analysis purposes, the mean data of lines A1, A2, and A3 were named TA processing, and the mean data of lines B1, B2, and B3 were named TB processing. 3 Results 3.1 Analysis of exogenous gene stability and expression in GM 107 The PCR amplification products of GM 107 from the test-forest are shown in Supplementary Figure 2. Using Cry1Ac and Cry3A specific primers, 546bp and 667bp bands were amplified from the six transgenic lines and positive plasmids. Additionally, 507bp bands were amplified from the A1, A2, and A3 lines and positive plasmids using BADH specific primers. In turn, 478bp bands were amplified from the B1, B2, and B3 lines and the positive plasmid using NTHK1 specific primers. No bands were amplified in the samples of non-transgenic poplar using the combination of the above primers, indicating that no loss of exogenous genes occurred during the field growth and development of GM 107. An enzyme-linked immunosorbent assay (ELISA) was used to detect toxins in the highly resistant lines A1 and B1. The results of these assays are summarized in Supplementary Figure 3. Toxic proteins were detected in the leaves, phloem, and roots of A1 and B1lines. The Cry1Ac content was much lower than that of Cry3A, and the difference in Cry1Ac was small in the A1 and B1 lines, whereas the difference in Cry3A was large, and the difference in the B1 line was much higher than that of A1. Additionally, Cry1Ac content was highest in the long and short branches and leaves, followed by the phloem, and lowest in the roots, with significant differences among these organs. Similarly, Cry3A content changes differed in A1 and B1 lines; the expression orders were leaf > phloem > root in the A1 line and phloem > leaf > root in the B1 line. 3.2 Arthropod community analysis of GM 107 experimental forest The survey results of seven lines in the test-forest were used as the sampling times, and 35 samples were collected over three years. The dilution curve showed that the species accumulation curves of several surveys tended to be smooth (Supplementary Figure 4), indicating that the survey results fully reflected the arthropod community at GM 107. Figure 2a-c shows the composition of the arthropod communities at the order level in the Mancheng test-forest for three consecutive years. In 2018, 10485 arthropods belonging to two classes, 10 orders, 48 families, and 71 species were counted; meanwhile, in 2019, 15077 arthropods belonging to three classes, nine orders, 61 families, and 90 species were counted; and in 2020, 8930 arthropods belonging to three classes, 10 orders, 56 families, and 73 species were counted. From the perspective of species richness, the number of species in 2019 and 2020 slightly increased, compared with that in 2018, which may be related to the growth stage of poplars. From the perspective of quantity, Hemiptera, Lepidoptera, and Hymenoptera accounted for a higher proportion, and there were varying degrees of fluctuation over the past three years without an obvious pattern of change. Figure 2d-f shows the composition of arthropod communities at the order level in the experimental forest over different years. In 2018, 5538 arthropods belonging to two classes, nine orders, 49 families, and 64 species were investigated in Yanshan, including 4015 insects and 1523 arachnids. A total of 13098 arthropods belonging to two classes, 10 orders, 50 families, and 68 species were investigated in Luannan, including 12185 insects and 913 spiders, all of which were dominated by lepidopterans. Significant differences in abundance in the test-forest at the order level in different regions were observed. For example, the relative abundances of Lepidoptera were 33.30% in Mancheng, 36.29% in Yanshan, and 45.83% in Luannan. Meanwhile, the relative abundances of Hemiptera were 18.37% in Mancheng, 5.70% in Yanshan, and 24.91% in Luannan. In turn, the relative abundances of Hymenoptera were 31.66%, 15.20%, and 9.47%, in Mancheng, Yanshan, and Luannan, respectively, indicating that the arthropod community compositions of GM 107 at the different sites were significantly different. 3.3 Comparison of arthropod communities in different lines of GM 107 test-forests A Venn diagram shows the species composition of GM 107 (Figure 3a). Overall, transgenic lines and controls exhibited similar species compositions and community structures. In 2018 and 2019, 53 species of control lines were investigated, with the number of genetically modified lines ranging from 49 to 55 and 35 species shared by seven lines. In 2020, 51 species were investigated in the control line and the number of genetically modified lines ranged from 42 to 53, with 28 common species in seven lines. The relative abundance of species fluctuated among lines; however, the overall community structure was similar. Figure 3b shows the quantitative structure of the arthropod communities at the order level. Overall, the number of insect populations on the transgenic lines in the three test stands was lower than that in the control. In addition, although relatively small, differences in the number of insect populations in each transgenic line were observed among them. As shown in Figure 3c, the number of species in each line of Mancheng ranged from 49 to 54, with 32 common species. Additionally, the number of species in line B2 was the highest and those in lines A3 and B3 were the lowest. In turn, the number of species in each line of the Yanshan area ranged from 41 to 50, with 31 common species. In this case, line B2 showed the highest number of species and A1 the lowest. Lastly, the number of species in Luannan ranged from 52 to 67, with 34 common species. In this test-forest, A2 and B2 showed the highest and lowest number of species, respectively. Most arthropod species appeared in both transgenic and control lines, and the number of species fluctuated among lines; however, it was similar between the transgenic and control lines, and there was no significant change. Figure 2d shows the quantitative structure of the arthropod communities at the order level. Overall, the number of insect populations in the transgenic lines in the three test-stands was smaller than that in control stands and differences in the number of insect population among transgenic lines was relatively small. 3.4 Community characteristic index of GM 107 lines Diversity reflects community richness and variation. Figure 4a,c,e shows that during 2018-2020, the Shannon diversity index value for each GM 107 line was 2.48-2.68, 2.40-2.64, and 2.60-2.83, respectively. In turn, the Pielou evenness index was 0.69-0.73, 0.67-0.72, and 0.74-0.79, respectively, and the Berger-Parker dominance index was 0.25-0.32, 0.26-0.36, and 0.16-0.28, respectively. These results indicate that, over time, the arthropod diversity and evenness index increased, the dominance index decreased, and community stability increased. Alternatively, the data might suggest that the arthropod community system was not completely established in the early stages of GM 107 establishment, and the arthropod community tended to remain stable as the trees in the test stands grew. A comparison of the community characteristic indices of different lines showed that there was no significant difference between the group of transgenic lines and the control group over three consecutive years. The Shannon-diversity index values of GM 107 lines in the experimental forests in MC, YS, and LN were 2.48-2.68, 2.63-2.89, and 2.48-2.79, respectively (Figure 4b, d, f). The Pielou evenness index values were 0.69-0.73, 0.77-0.82, and 0.69-0.77, respectively. Finally, the Berger-Parker dominance index values were 0.25-0.32, 0.17-0.24 and 0.18-0.32, respectively. The results of the community characteristic indices of the different sites showed that the community stability at the different sites was as follows: YS > LN > MC. In the test forests in MC and YS there was no significant difference between the group of transgenic lines and the control group. However, the diversity and evenness index values of the transgenic lines in the LN test forest increased whereas dominance decreased, indicating that the transgenic poplar arthropod community in the LN test forest was more stable. The largest number of lepidopterans occurred in the experimental forest in LN, indicating that the transgenic lines at the nodes of the lepidopteran outbreak were beneficial for community stability. 3.5 Analysis of insect resistance of GM 107 lines The occurrence of the target insects is shown in Figure 5. From 2018 to 2020, the number of lepidopteran insects in the MC test forest was stable, and the number of insect populations were 3942, 3380, and 3600, respectively. Populations were mainly composed of Lyonetiidae, Tortricidae, Arctiidae, and Notodontidae, and were dominated by Lyonetiidae. After planting, the number of insects belonging to Tortricidae gradually decreased, and the numbers of insects belonging to Arctiidae and Notodontidae showed an outbreak trend every other year. In turn, the number of insects in the transgenic lines was lower than that in the control line for three consecutive years (a significant difference in 2018), and the number of Lepidopteridae was not significantly different among the transgenic lines. In turn, coleopteran insects showed an increasing trend each year, and the number of insects was 262, 1678 and 2814, respectively, and mainly comprise Chrysomelidae and Coccinellidae. The number of Coleoptera insects on the transgenic lines in 2018 and 2020 was lower than that on the control, and there was no significant difference in 2019. Insects belonging to Lepidopteridae differed significantly among the three sites. Thus, Tortricidae and Phylloscopidae were dominant in the forest in MC. Meanwhile, Phylloscopidae, Tortricidae, and Notodontidae were dominant in the forest in YS, and Arctiidae and Tortricidae were dominant in the forest in LN. The composition of Coleoptera families was similar in the three experimental forests, and they were mainly composed of Chrysomelidae, Coccinellidae, and Carabidae, with Chrysomelidae being dominant. The transgenic lines mainly inhibited insects belonging to Chrysomelidae but had no significant effect on insects belonging to Coccinellidae and Carabidae. The populations of Lepidoptera and Coleoptera in the transgenic lines were significantly smaller than those in the CK in all three test forests. Principal response curve (PRC) analysis is applicable to the study of dynamic community changes. Table 1 shows that the first model axis of each survey group was highly representative, and the Monte Carlo test reached significance (except in 2019). These results indicate that the first model axis of the three test-forests was highly representative, and subsequent PRC analysis based on the first model axis was more reliable. With respect to the arthropod community differences between transgenic poplars and the control, seasonal factors accounted for 62.08%, 70.73%, and 61.57%, of the variance respectively, and transgenic events accounted for 14.54%, 5.63%, and 10.68%. Seasonal factors in the forests in YS and LN explained 46.36% and 40.49% of the variance, respectively, and transgene events explained 26.30% and 21.38%, indicating that seasonal factors consistently had a greater effect on poplar arthropod communities than transgene events. Figure 6 shows that, except for 2020, the two transgenic lines showed different degrees of negative deviation. The variation trend of the main response curve in different years differed and the deviation trend of the test-forest in different regions was relatively consistent. In all three experimental forests, the deviations in arthropod communities in transgenic lines were large in August, September, and October 2018. The deviations were most severe in the experimental forest in LN, whereas the differences were greatest in May in 2019 and 2020. The correlation between species weights and the main response curve in Figure 5 can be interpreted as the closeness (affinity) of each species to the chart. The figure shows species whose absolute values were greater than 0.1. Insects that responded strongly to the PRC curve (species weight > 0.4) in 2018 in the test-forest in MC included Arctiidae, Pyralidae, Lyonetiidae, and Limacodidae. Meanwhile, Thomisidae, Tortricidae, and Formicidae responded strongly to the PRC curve in 2019, whereas Aphididae responded strongly to the PRC curve in 2020 (species weight=1.449). The insects that responded strongly to the PRC curve in the forest in YS included Arctiidae, Chrysomelidae, Notodontidae, and Tortricidae, whereas those in the PRC curve in the forest in LN included Arctiidae, Chrysomelidae, and Notodontidae. Most of these families belong to Lepidoptera or Coleoptera, indicating that the poplar transgenic lines showed obvious anti-insect efficacy in the three test-forests. The weights of the other families were < 0.4. Notably, Coccinellidae responded to the PRC curve for three consecutive years, and the weight values in 2018, 2019, and 2020 were 0.338, 0.256, and 0.173, respectively. Thomisidae responded to the PRC curve across experimental forests in MC, YS, and LN, with weight values of 0.3136, 0.1075, and 0.1313, respectively. The PRC curves of the s in MC and YS simultaneously responded to Cicadellidae, with weights of 0.139 and 0.2156, respectively, while the PRC curves of the forests in MC and LN responded to Phylloxidae, with weights of 0.2309 and 0.2840, respectively. 3.6 Arthropod community nutrition-structure of GM 107 According to the classification of trophic layers and taxa, the nutrient layer richness and taxa composition of arthropods in GM 107 at different sites are shown in Supplementary Figure 5-7. The three test-forests showed the same pattern; that is, the rocky species richness of arthropod groups in TA and TB decreased compared with those in the control lines, whereas the middle and top species richness increased compared with those of the control. In the experimental forest in MC (Supplementary Figure 5), the rocky species richness of the TA and TB arthropod fauna was 0.5652 and 0.5738, respectively, which were 13.19% and 11.87% lower than that of the control (0.6511). In turn, the median species richness was 0.3597 and 0.3523, respectively, which was 20.83% and 18.34% higher than that of the control (0.2977). The top species richness was 0.0747 and 0.0738, which were 46.18% and 44.42 %, higher, respectively, compared with the control (0.0511). In the forest in YS (Supplementary Figure 6), the species richness of the TA and TB communities was 0.4887 and 0.4922, respectively, i.e., 30.21% and 29.71% lower, respectively, than that of the control (0.7002). In turn, the median species richness was 0.2755 and 0.2667, respectively, which was 82.78% and 88.83% higher than that of the control (0.1459). Lastly, the top species richness was 0.2451 and 0.2318, i.e., 59.26% and 50.62 % higher, respectively, that that of the control (0.1539). Meanwhile, in the test forest in LN (Supplementary Figure 7), the rocky fauna richness of TA and TB arthropods was 0.7733 and 0.5992, respectively, which was 12.82% and 32.45% lower than that of the control group (0.8870). The median species richness was 0.1695 and 0.1143, which was 93.94% and 30.78 % higher, respectively, than that of the control (0.0874). Finally, the top species richness was 0.0572 and 0.0367, i.e., 123.44% and 43.36 %, higher, respectively, than that of the control (0.0256). The dominant species determines the nature of the community, and when the dominant species change, it may change the structure and nature of the community to a certain extent. The top species were the dominant groups of Tarantula and Thomisids, among which the main species were Chinese tarantulas and Tritaphosa. In turn, the dominant groups of the median species were Heterogyna, Ladybird, Araneidae, Lacewings, and Aphid fly eats, among which the main species were black-brown ants, harlequin ladybirds, turtle ladybirds, and Chinese Caolinids. The composition and dominant groups of the TA and TB lines were basically the same as those of the control base, middle, and top species, and the dominance of the target groups and some other groups changed to some extent as well. For example, the number and dominance of Phytophthira and locust arthropods in TA and TB communities increased in the test forests of MC and YS compared to those in the control group. Lastly, the number of Araneidae in the arthropod communities of the TA and TB lines decreased but their dominance did not decrease significantly. Nutrient layer richness and group composition of arthropods in each line of the MC test forest in 2019 and 2020 are shown in Supplementary Figure 8 and Supplementary Figure 9. In 2019 (Supplementary Figure 8), the base species richness of the TA community increased by 2.65% compared with that of the control, whereas that of the TB community decreased by 10.26%, compared with that of the control. The median number of species in the TA community decreased by 3.13% and that in the TB community increased by 7.09%. The top species in TA, increased by 15.78%, and the arthropod community on TB decreased by 4.75%. In terms of trophic groups, the composition and dominant groups on transgenic lines and controls were essentially the same, and the dominance degree and number of some groups showed the same trend. For example, in the base species, the number of lepidopteran insect pests in the communities on TA and TB decreased, and their dominance decreased by 4.59% and 20.48%, respectively, compared with the control. In turn, the number of sawflies and their dominance increased by 171.43% and 100%, respectively, compared to the control. Further, compared to the control group, the number of heteropterans and their dominance increased by 31.62% and 46.15%, respectively, relative to the control. In contrast, the number of locusts and their dominance increased by 114.29% compared to the control. Conversely, the number of Phyllostraca species and their dominance decreased by 32.00% and 36.00%, respectively but the number of leafhoppers and their dominance increased by 136.54% and 65.38%, respectively. Further, the number of median species and their dominance on TA and TB increased by 75.93% and 85.19%, respectively. In particular, the number of dwarf spiders and their dominance increased by 67.21% and 31.15%, respectively; the number of ladybirds and their dominance decreased by 13.48% and 22.38%, respectively. Meanwhile, the number of top species and their dominance on TA and TB lines increased by 129.51% and 70.49%, respectively. In 2020 (Supplementary Figure 9), compared to the control, the basal and top species richness of the transgenic lines decreased, whereas the median species richness increased. Specifically, the base species decreased by 5.91% and 4.48%, top species decreased by 29.73% and 8.11%, and median species increased by 21.36% and 15.95%, respectively. In terms of the abundance and quantity of trophic groups, the number of lepidopterans in TA and TB arthropod communities decreased by 19.54% and 34.82%, those of leafhoppers increased by 75.84% and 63.91%, respectively, and those of Phyllostraca decreased by 25.71% and 31.67%, respectively. The number of Heterogyna species and their dominance increased by 51.70% and 109.22%, respectively. Among the median species, the abundance of lacewings in TA and TB increased by 54.55% and 44.81%, respectively; the abundance of dwarf spiders increased by 67.53% and 83.12%, respectively; and the abundance of thomisids decreased by 45.95% and 8.11%, respectively. 3.7 Functional groups and quantitative structure of arthropod community in GM 107 forest The composition of various groups in the GM 107 forests reflects the complexity of the food network and indicates the extent of pest control. In terms of feeding habits, the abundance of hyphae was the highest and that of parasites was the lowest. There were some differences in the composition and structure of the functional groups between the different years and locations. Compared to the control, the abundance of phytophages decreased, the relative abundance of neutrals increased, and the differences between predatory insects, spiders, and parasites were relatively small (Figure 7a and 7b). In terms of community stability indices, S s /S t , S n /S p , and S a /S b fluctuated greatly among different years and sites in the test forests (Figure 8c and 8d). Particularly, S s /S t of the transgenic lines was higher than that of the control and S a /S b was lower than that of the control, indicating that the transgenic lines had a stronger interspecific-restriction effect and higher, natural damage-control efficiency upon arthropod attack. 3.8 Arthropod community structure of GM 107 Non-metric multidimensional scaling (NMDS) analysis was performed on the arthropod communities on GM 107 lines in different years (Figure 8a). In 2018 and 2019, the arthropod communities in the experimental forest in MC were concentrated in the second and third quadrants, respectively, while in 2020 they were concentrated in the first and fourth quadrants. In the same year, transgenic and control lines could not be distinguished on the first and second axes. The results showed (Figure 9c) that the community was clustered into four groups when λ=0.5. The control and B1 lines, and other lines clustered together in 2018; and 2019 and 2020 were respectively cluster. According to the NMDS of the community structure of the experimental forests in different regions (Figure 8b), the experimental forests in MC and YS were distributed in the third and first quadrants, respectively, whereas the experimental forests in LN were distributed in the third and fourth quadrants. There was a certain degree of deviation between the transgenic and control in all three test stands, which was smaller than the community distance between the test stands. The results of cluster analysis (Figure 9d) were similar to those of the NMDS. This indicated that the arthropod community structure of GM 107 lines was different from that of the control, and the difference was smaller than that among years and planting sites. 4 Discussion The commercialization of genetically modified trees in China is slow, mainly because of ecological safety issues (Valenzuela et al., 2006). The expression of exogenous genes may affect the secretion of nutrients and some secondary metabolites in host plants and play a key role in the feeding of phytophagous insects and natural predators, as well as the host-seeking and egg-laying of insects, thus affecting the arthropod community and even the entire ecosystem functionality and service provision (Schultz, 1988; Turlings and Erb, 2018; Noushahi and Hussain, 2020). Poplar has a long growth cycle, and leaf-eating and stinging insects are directly exposed to the insecticidal protein in Bt transgenic poplar during the entire growth process. Therefore, the impact of transgenic 107 poplar on arthropod communities is key to its ecological safety evaluation. Research has been conducted to assess the safety of genetically modified crops (Ou et al., 2015). Crop insect-resistance plays a major role in insect control, and the results of the assessment of its ecological safety should be a measure of its impact on agricultural ecosystems. Woody plants are often dominant in ecosystems, and their ecological risk assessment is complicated by longevity issues and the difficulty of extrapolating results from small-scale studies to large-scale plantations (Pons et al., 2005; Lu et al., 2010; Dhurua and Gujar, 2011; Bai et al., 2012; Wan et al., 2012; Fabrick et al., 2014). Comprehensive and reliable data can only be obtained through relatively long-term systematic monitoring of genetically modified trees that are released for use in the field. In previous studies, Bt transgenic poplar trees showed good and stable insect control under field conditions but their toxic effects on different target pests varied (Valenzuela et al., 2022). For example, the toxic effects of Cry1Ac on the four Lepidoptera species were as follows: Micromelalopha troglodyta > Hyphantria cunea > Clostera anachoreta > Lymantria dispar , and the toxic effects of Cry3A on Coleoptera were as follows: Plagiodera versicolora > Cerambycidae (Huang et al., 2021). By monitoring target arthropod communities in different regions in the same year and in the same region over three consecutive years, this study found that differences in the sensitivity of different geographical arthropod populations on GM 107 to insecticidal proteins. However, this difference was within the range of natural differences, and the target insect was still sensitive. Additionally, we found that GM 107 shows anti-insect selectivity and that the degree of toxicity to different species of Lepidoptera or Coleoptera varies. With respect to lepidopteran insects with high occurrence rates in the field, their resistance to the tree toxin was as follows: Lamphidae > Canopidae > Borellidae > Leaf stealers > Acanthidae. As for Coleoptera, the effect on Phyllopteridae was strong but weak on ladybirds and beetles. The arthropod community of GM107 was significantly different from that of the control group; however, there were no significant differences among the different transgenic lines. One reason for this may be the small number of target insects occurring during the research. Alternatively, the differences between the field environment and that in the laboratory, and the resistance of the high-resistance transgenic line being lower in the field than indoors may explain the lack of significance of the differences in arthropod communities among transgenic lines. The occurrence of agricultural and forestry insect pests in the field environment is often characterized by suddenness, randomness, diversity, and non-uniformity, and is affected by cultivation mode, climatic conditions (especially spring temperature and rainfall), regional factors and other factors (Fang et al., 1997).Therefore, the effects of transgenic plants on non-target insects and natural enemies are inconsistent. In a study of Bt transgenic crops, most researchers found that Bt toxic proteins have a direct killing effect neither on non-target pests nor on natural enemies, but may have indirect effects on the ecosystem due to changes in intermediate competition or host malnutrition (Catarino et al., 2015). Studies have shown that transferring Cry3A to potatoes has no adverse effects on non-target pests that may come into contact with the crop (Guan et al., 2018). The number of beneficial arthropods in the plots where Bt toxin was sprayed on the leaves was much higher than that in the plots where traditional chemical pesticides were used. In the areas where genetically modified potatoes were grown, aphids were controlled by natural predators alone, whereas aphid populations increased in plots treated with conventional chemical insecticides (non-aphid insecticides). Wang et al. (2003) conducted spot and field surveys of Bt transgenic cotton and conventional cotton fields and found that the larval stock of Bt transgenic cotton was significantly reduced, and the development of the red spider population was faster than that observed in conventional cotton fields (Mendelsohn et al., 2003). Our results differed from those of previous studies. We did not observe any significant difference between species richness of GM 107 and that of the control group. Further, there was no increase in the number of non-target pests or natural enemies, and the relative abundance of natural enemies and neutral insects in the community increased. The relative number of natural enemies reflects community control efficiency. The food sources of natural enemies are not only dependent on pests. Neutral insects are also an important food source for predatory natural enemies, and maintaining a large population of neutral insects plays an important regulatory role in the development of predatory natural enemies, and even the whole arthropod community (Pons et al., 2005). Therefore, GM 107 not only did not increase the number of non-target insects but, additionally, it optimized the composition of the arthropod community and enhanced ecological immunity. Changes in plant traits induced by the introduction of exogenous genes may change a community structure through interspecific relationships, such as food webs. In this study, we systematically evaluated the arthropod communities of two GM 107 types from the perspectives of communities, subcommunities, functional taxa, trophic layers, and functional groups. Although the arthropod community structure of GM 107 differed with growing environment and year, the community structures of the transgenic and control lines were similar. Many characteristics of the community reflected that the community diversity and evenness indices of transgenic lines were higher, whereas the dominance index was lower in some months, and when an outbreak of lepidopteran pests was severe. In terms of the seasonal dynamics of the overall community-characteristic index, the seasonal dynamics of the total community of GM 107 was relatively stable, presumably because of the strong pest resistance of the transgenic poplar forests, where no pest outbreaks occurred. Other community indices also reflected that the arthropod community-nutrition structure of GM 107 was more sustainable than that of the control group; specifically, there were fewer phytophagous insects and neutral arthropods, and the median and apex species increased, showing a beneficial ecological effect. 5 Conclusion The exogenous gene of GM 107 plants was stable in the field, the toxic protein was expressed throughout the growing season, and the expression levels of toxic proteins were significantly different in different Bt types and different tree organs. The changes in the GM 107 population were complex, and the arthropod community characteristic index was relatively stable. When a lepidopteran pest outbreak was severe, the community stability of the transgenic lines was higher than that of the control plants but in the absence of major pest outbreaks in the community, the community stability of the transgenic lines did not significantly differ from that of the control plants. In the field, GM 107 showed stable insecticidal effects against target pests and insect resistance selectivity, but had no inhibitory or proliferative effects on natural enemies or neutral insect populations. GM 107 effectively showed an increased ability for natural pest control and stability of the arthropod community. Furthermore, the arthropod community structure on GM 107 was similar to that on the control trees . I n this study, the field trial of transgenic Populus × euramericana '107' was conducted on a limited scale with relatively short monitoring periods. Therefore, further investigations into arthropod communities within these transgenic poplar plantations should involve expanded and continuous monitoring. Declarations Ethics approval and consent to participate Ehics, Consent to Participate, and Consent to Publish declarations: not applicable. Consent for publication Not applicable Availability of data and materials Data will be available on request and were provided within figures of the manuscript or supplementary information files. Competing interests The authors declare that they have no competing interests" in this section. Funding This study was supported by the Major Project of Agricultural Biological Breeding (2022ZD0401502), the Provincial Key Research and Development Program of Hebei (21326301D), the Natural Science Foundation of Hebei Province (C2023204098) and the Young Elite Scientists Sponsorship Program by CAST (2023QNRC001) Authors’ contributions Yali Huang: Investigation, Data curation, Performed statistical analysis and visualization, Writing - original draft. Zijie Zhang , Shijie Wang: Statistical analysis and visualization. JinMao Wang , Weizhen Zhang, Chengcheng Li: Review the manuscript. Minsheng Yang: Designed the study, Writing - Review & Editing. All authors read and approved the final manuscript. Acknowledgment We would like to thank the District Nursery Field for providing the planting site, as well as the editor and reviewers for valuable suggestions to improve the previous version of the manuscript. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: https://scientific-publishing.webshop.elsevier.com/ References Bai, Y.Y., Yan, R.H., Ye, G.Y., et al. 2012, Field response of aboveground non-target arthropod community to transgenic Bt-Cry1Ab rice plant residues in postharvest seasons. Transgenic Research, 21(5): 1023-1032. doi: 10.1007/s11248-012-9590-6. Blondel, J., 2003. Guilds or functional groups: Does it matter?. 100(2): 223-231. Camargo, A.M., Andow, D.A., Castañera, P., et al. 2018. First detection of a Sesamia nonagrioides resistance allele to Bt maize in Europe. Science Reports, 8(1): 3977. doi:10.1038/s41598-018-21943-4 Catarino, R., Ceddia, G., Areal, F.J., et al. 2015. The impact of secondary pests on Bacillus thuringiensis (Bt) crops. Plant Biotechnology Journal, 13(5): 601-612. doi:10.1111/pbi.12363. Chandrasena, D.I., Signorini, A.M., Abratti, G., et al. 2018. Characterization of field-evolved resistance to Bacillus thuringiensis-derived Cry1F δ-endotoxin in Spodoptera frugiperda populations from Argentina. Pest management science, 74(3): 746-754. doi:10.1002/ps.4776 Dhurua, S., Gujar, G.T., 2011. Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India. Pest Management Science, 67(8): 898-903.doi: 10.1002/ps.2127 Fabrick, J.A., Ponnuraj, J., Singh, A., et al. 2014, Alternative splicing and highly variable cadherin transcripts associated with field-evolved resistance of pink bollworm to Bt cotton in India. PloS One, 9(5): e97900. doi: 10.1371/journal.pone.0097900 Fang, L.L., Zhou, C., Wang R., et al. 2019. RCS Feature-aided insect target tracking algorithm. journal of radars, 8(05):598-605. doi:10.1030.TN.20191011.1730.002 Ferré, J., Van Rie, J., 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annual review of entomology, 47: 501-533. doi:10.1146/annurev.ento.47.091201.145234 Fu W., Tang T., Wang P., et al., 2022, Resistance Development of Four Bt Insecticidal Crystal Proteins against Btk-resistant Strain of Plutella xylostella in the Laboratory. Chinese Journal of Biological Control, 38(02):328-332. doi:10.16409/j.cnki.2095-039x.2021.05.019. Guan, Z.J., Lu, B.S., Huo, Y.L., et al., 2018. Effects of Bt crops on non-target insect pests. Biodiversity Science, 26(06): 636-644. doi:10.17520/biods.2017315 Huang, Y., Zhen, Z., Cui, Z., et al. 2021. Growth and arthropod community characteristics of transgenic poplar 741 in an experimental forest. Industrial Crops and Products, 162: 113284. doi:10.1016/j.indcrop.2021.113284 Hu, J.J., Yang, M.S., Lu, M.Z., 2010. Advances in biosafety studies on transgenic insect-resistant poplars in China. Biodiversity Science. 18(04): 336-345. doi:10.3724/SP.J.1003.2010.336 Juliano, R.F., David, A., Andow, b., et al. 2014. Field-evolved resistance to Cry1F maize by Spodoptera frugiperda (Lepidoptera: Noctuidae) in Brazil-ScienceDirect. Crop Protection, 64(3): 150-158. doi:10.1016/j.cropro.2014.06.019 Liliana, P.L., Mario, S., Alejandra, B., 2012. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. Fems Microbiology Reviews, 37(1):3-22. doi:10.1111/j.1574-6976.2012.00341.x. Liu, D.Y., Zhang, J., Dong, Y., et al., 2016. Genetic transformation and expression of Cry1Ac–Cry3A–NTHK1 genes in Populus × euramericana "Neva". Acta Physiologiae Plantarum, 2016, 38(7): 1-11. doi:10.1007/s11738-016-2195-6 Lu, Y., Wu, K., Jiang, Y., et al. 2010, Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China. Science, 328(5982): 1151-1154. doi: 10.1126/science.1187881 Martinez, J.C., Caprio, M.A., Friedenberg, N.A., 2018. Density dependence and growth rate: evolutionary effects on resistance development to Bt ( Bacillus thuringiensis ). Journal of economic entomology. 111(1): 382-390. doi: 10.1093/jee/tox323// Marroquin, L.D., Elyassnia, D., Griffitts, J.S., et al., 2000. Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. Genetics, 155(4): 1693-1699. doi:10.1093/genetics/155.4.1693 Meilan, R., Ma, C., Cheng, S., et al. 2000. High levels of Roundup and leaf-beetle resistance in genetically engineered hybrid cottonwoods. Plant Physiology Mendelsohn, M., Kough, J., Vaituzis, Z., et al., 2003. Are Bt crops safe?. Nature Biotechnology, 21(9): 003-1009. doi: 10.1038/nbt0903-1003 Noushahi, H. A., Hussain, M., 2020. Risk assessment of genetically modified plants: a review. Asian Journal of Research in Crop Science. 1-10. doi:10.9734/ajrcs/2020/v5i330095 Pons, X., Lumbierres, B., Lopez, C., et al. 2005, Abundance of non-target pests in transgenic Bt-maize: A farm scale study. European Journal of Entomology, 102(1): 73-79 Storer, N.P, Kubiszak, M.E., King, J.E., et al., 2012. Status of resistance to Bt maize in Spodoptera frugiperda: Lessons from Puerto Rico. Journal of Invertebrate Pathology. 110(3): 294-300. doi:10.1016/j.jip.2012.04.007 Tabashnik, B.E., Brévault, T., Carrière, Y., 2013. Insect resistance to Bet crops: lessons from the first billion acres. Nature biotechnology, 31(6): 510-521. doi:10.1038/nbt.2597 Tabashnik, B.E., Cushing, N.L., Naomi, F., et al. 1990. Field Development of Resistance to Bacillus thuringiensis in Diamondback Moth (Lepidoptera: Plutellidae). J.econ.entomol, 83(5): 1671-1676. doi:10.1016/0020-7322(90)90018-K Tabashnik, B.E., Carrière, Y., 2017. Surge in insect resistance to transgenic crops and prospects for sustainability. Nature biotechnology, 35(10): 926-935. doi:10.1038/nbt.3974 Turlings, T.C.J., Erb, M., 2018. Tritrophic interactions mediated by herbivore-induced plant volatiles: Mechanisms, ecological relevance, and application potential. Annual Review of Entomology, 63: 433-452. doi:10.1146/annurev-ento-020117-043507 Valenzuela, S., Balocchi, C., Rodríguez, J., 2006. Transgenic trees and forestry biosafety. Electronic Journal of Biotechnology, 9(3): 335-339. doi:10.2225/vol9-issue3-fulltext-22 Wang, B., Li, H.M., Cao, H.Q., et al. Mechanisms and Applications of Plant-Herbivore-Natural Enemy Tritrophic Interactions Mediated by Volatile Organic Compounds. Scientia Agricultura Sinica, 2021, 54(08): 1653-1672. doi: 10.3864/j.issn.0578-1752.2021.08.007 Wang, Y.X., Hao, Y.S., Du, J.Z., et al., 2014. Recovery of transgenic Populus plants with modified Bt Cry1Ac gene and their insect-resistance assay. Chinese Agricultural Science Bulletin, 30(28): 23-28. Wan, P., Huang, Y., Wu, H., et al. 2012, Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China. PLoS One, 7(1): e29975. doi: 10.1371/journal.pone.0029975 Yang, M.S., Gao, B.J., Wang, J.M., et al., 2005. Analysis of Main Characteristic of White Poplar Hybrid 741 Transformed Two Insect-Resistant Genes. Scientia silvae sinicae, 2005, 41(01): 91-97. Yang, R.L., Wang, A.X., Zhang, J., et al. 2016. Genetic transformation and expression of transgenic lines of Populus × euramericana with insect-resistance and salt-tolerance genes. Genetics and Molecular Research : GMR, 15(2): 248-249. doi: 10.4238/gmr.15028635 Zhang, C., Wei, J., Naing, Z.L., et al. 2021. Endogenous serpin reduces toxicity of Bacillus thuringiensis Cry1Ac against Helicoverpa armigera (Hübner). Pesticide biochemistry and physiology, 175: 104837. doi:10.1016/j.pestbp.2021.104837 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.png SupplementaryFigure2.png SupplementaryFigure3.png SupplementaryFigure4.png SupplementaryFigure5.png SupplementaryFigure6.png SupplementaryFigure7.png SupplementaryFigure8.png SupplementaryFigure9.png Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 30 Sep, 2025 Reviews received at journal 06 Aug, 2025 Reviewers agreed at journal 21 Jul, 2025 Reviews received at journal 20 Jul, 2025 Reviewers agreed at journal 17 Jul, 2025 Reviewers agreed at journal 15 Jul, 2025 Reviewers invited by journal 15 Jul, 2025 Editor assigned by journal 15 Jul, 2025 Editor invited by journal 09 Jul, 2025 Submission checks completed at journal 07 Jul, 2025 First submitted to journal 07 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6954149","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486515427,"identity":"9bd4d072-d114-4424-b226-72c4c420fd33","order_by":0,"name":"Yali Huang","email":"","orcid":"","institution":"Chengdu University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yali","middleName":"","lastName":"Huang","suffix":""},{"id":486515428,"identity":"47b6ec54-4232-40f6-a0bf-682592a9ec4e","order_by":1,"name":"Zijie Zhang","email":"","orcid":"","institution":"Forestry College, Hebei Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Zijie","middleName":"","lastName":"Zhang","suffix":""},{"id":486515429,"identity":"60c037ec-d2db-4135-89c2-098617f52964","order_by":2,"name":"ChengCheng Li","email":"","orcid":"","institution":"Center of Education and Communications of Ecology and Environment of Sichuan Province","correspondingAuthor":false,"prefix":"","firstName":"ChengCheng","middleName":"","lastName":"Li","suffix":""},{"id":486515430,"identity":"30d28355-42ab-4108-b896-f1fd7e801a9c","order_by":3,"name":"Shijie Wang","email":"","orcid":"","institution":"Forestry College, Hebei Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Shijie","middleName":"","lastName":"Wang","suffix":""},{"id":486515431,"identity":"f09081cd-68c3-476f-bc72-bf62a0db9f31","order_by":4,"name":"WeiZhen Zhang","email":"","orcid":"","institution":"Chengdu University of Technology","correspondingAuthor":false,"prefix":"","firstName":"WeiZhen","middleName":"","lastName":"Zhang","suffix":""},{"id":486515432,"identity":"a59aecd1-0998-4d8d-9754-b9e917248e31","order_by":5,"name":"Jinmao Wang","email":"","orcid":"","institution":"Forestry College, Hebei Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Jinmao","middleName":"","lastName":"Wang","suffix":""},{"id":486515433,"identity":"c21d4db7-5a44-4053-872d-5971a7f6625f","order_by":6,"name":"Minsheng Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYPACCR429sbGBx9I0GIhx89zuNlwBglaKowlZ6S3SXMQo9bg+NnDr278kUjccPNhgzQDg52cbgMhLWfy0qxz24Babic2GBcwJBubHSCk5UCOmXFuA0RL8gyGA4nbCGo5/8bMOAfssIMNh3mI0nIjx/hxDpsE0PuMjc1EaZG88caMGegXYCAnNjPOMCDCL3znc4w/5/ypA0bl8ec/PlTYyRHUonCAgU0CyZ0ElIOAfAMDMynJZBSMglEwCkYiAACyTEgdO8UNyQAAAABJRU5ErkJggg==","orcid":"","institution":"Forestry College, Hebei Agriculture University","correspondingAuthor":true,"prefix":"","firstName":"Minsheng","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-06-23 07:54:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6954149/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6954149/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07798-8","type":"published","date":"2025-12-01T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86939036,"identity":"cc52f148-a3b2-454d-8954-3c9bc6b94e2b","added_by":"auto","created_at":"2025-07-17 11:23:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5036037,"visible":true,"origin":"","legend":"\u003cp\u003ePlanting area of the GM 107 experimental forests.\u003cstrong\u003e a,\u003c/strong\u003e Geographical locations of the three test sites. \u003cstrong\u003eb,\u003c/strong\u003e Photograph of the Mancheng test forest.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/71a36834f041386369adf5c4.png"},{"id":86939041,"identity":"062bde2a-c8d3-49d7-87a8-a6b92038391f","added_by":"auto","created_at":"2025-07-17 11:23:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":593500,"visible":true,"origin":"","legend":"\u003cp\u003eArthropod community composition of GM 107 in the experimental forests. \u003cstrong\u003ea-c,\u003c/strong\u003eArthropod community compositions in different years in Mancheng; \u003cstrong\u003ed-f,\u003c/strong\u003eArthropod compositions in different regions in 2018. MC: Mancheng; YS: Yanshan; LN: Luannan. Values within brackets are number of families (NF) and number of species (NS), respectively.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/4875e807e07394d39a62ef69.png"},{"id":86939037,"identity":"224c06ba-1f91-46a9-b759-fb25b63dbde7","added_by":"auto","created_at":"2025-07-17 11:23:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6596996,"visible":true,"origin":"","legend":"\u003cp\u003eArthropod community composition of GM 107 lines in different regions and years. \u003cstrong\u003ea-b\u003c/strong\u003e, Venn diagram and accumulation column diagram of arthropod community composition of GM 107 lines in different years; \u003cstrong\u003ec-d\u003c/strong\u003e, Venn diagram and accumulation column diagram of arthropod community composition of GM 107 lines in different regions.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/c578a600d5764ebafc64a662.png"},{"id":86939048,"identity":"d077d904-cbaf-4e0b-88ec-0f577b3abd22","added_by":"auto","created_at":"2025-07-17 11:23:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":433855,"visible":true,"origin":"","legend":"\u003cp\u003eCharacter index of arthropod communities in GM 107 ecosystems. Panels \u003cstrong\u003ea\u003c/strong\u003e, and \u003cstrong\u003ee\u003c/strong\u003e show the arthropod community-characteristics index in 2018, 2019, and 2020 in the experimental forest in Mancheng respectively. Panels \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, and \u003cstrong\u003ef\u003c/strong\u003e represent the arthropod community characteristics index in the experimental forests in Mancheng (MC), Yanshan (YS) and Luannan (LN).\u003c/p\u003e\n\u003cp\u003eNote，H, E and D represent the Shannon-Winner diversity index, Pielou evenness index, and Berger-parker dominance index, respectively.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/5dc93af1c2b40a8d56e8116c.png"},{"id":86939054,"identity":"945e4348-be94-418a-a2a7-aeac7247ba1e","added_by":"auto","created_at":"2025-07-17 11:23:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6387029,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of individual number of target insects in GM 107 ecosystems. \u003cstrong\u003ea,\u003c/strong\u003e Comparison of individuals of Lepidoptera in GM 107 in different locations. \u003cstrong\u003eb,\u003c/strong\u003e Comparison of individuals of Coleoptera in GM 107 in different locations. \u003cstrong\u003ec\u003c/strong\u003e, Comparison of individuals of Lepidoptera in GM 107 in different years. \u003cstrong\u003ed\u003c/strong\u003e, Comparison of individuals of Coleoptera in GM 107 in different years.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/798cbff6d8feebfe15f37f20.png"},{"id":86939050,"identity":"82a18729-0d07-4c22-ac5c-0ead8b3b9c99","added_by":"auto","created_at":"2025-07-17 11:23:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5857018,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of transgenic insect-resistant poplar on arthropods of different families. Panels \u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, and \u003cstrong\u003ec\u003c/strong\u003e show the Canonical coefficient of Mancheng (MC) in 2018 (a), 2019 (b) and 2020 (c); \u003cstrong\u003ed \u003c/strong\u003eand \u003cstrong\u003ee \u003c/strong\u003erepresent the Canonical coefficient of Yanshan (YS) and Luannan (LN) in 2018. The species weights shown on the right part of the diagram represent the affinity of each species with the response shown in the diagram. For the sake of clarity, only species with a weight larger than 0.1 or smaller than −0.1 are shown.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/9e422e01834f6663053702d0.png"},{"id":86939631,"identity":"2aa30bef-57f3-4d99-8857-5dcaa47f4ee5","added_by":"auto","created_at":"2025-07-17 11:31:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":539251,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional group structure and stability of arthropod community in GM 107 ecosystems. Panels a and b represent the functional group structure of arthropod community in GM 107 test-forests in different years and regions; panels c and d represent the arthropod community stability of GM 107 test forest in different years and regions.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/973f1d283620abde4754f6af.png"},{"id":86939060,"identity":"91825523-b3e7-4f2e-aaeb-86277e7e3d9f","added_by":"auto","created_at":"2025-07-17 11:23:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":4077532,"visible":true,"origin":"","legend":"\u003cp\u003eSimilarity analysis of arthropod community in GM 107.\u003c/p\u003e\n\u003cp\u003ea and b represent non-metric multidimensional scaling analysis of GM 107 arthropod communities in different years (a) and regions (b); c and d represent cluster analysis based on the Euclidean distance of GM 107 arthropod communities in different years (c) and regions (d).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/0dcd2cd47e5cfee2d806e12d.png"},{"id":97723890,"identity":"38008886-6803-44f6-919f-9d77e2a5ff7d","added_by":"auto","created_at":"2025-12-08 16:09:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29196491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/6d8a9069-354d-430c-8135-4e54832b1f8e.pdf"},{"id":86940655,"identity":"de2961bf-011f-4a5c-8a25-fd188abcad95","added_by":"auto","created_at":"2025-07-17 11:47:51","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":141070,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/6e70f2f9d59e318b76649a5f.png"},{"id":86939040,"identity":"a2841f99-8935-4088-99ed-4a7a2a30e3f5","added_by":"auto","created_at":"2025-07-17 11:23:51","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2033276,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/229cf7322d0df9d83cd562a5.png"},{"id":86939613,"identity":"9f7eb047-1bb0-4d16-b2d2-cfcb764f695a","added_by":"auto","created_at":"2025-07-17 11:31:52","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":67876,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/b5ec690d33f87598606935bd.png"},{"id":86939045,"identity":"4f927530-b9ff-4b94-8e8c-3aaa50b7a0b4","added_by":"auto","created_at":"2025-07-17 11:23:51","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1657745,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/fe4e5e00b59f33c748135f3c.png"},{"id":86939625,"identity":"931b9273-e049-4280-a134-1df1b8fc1179","added_by":"auto","created_at":"2025-07-17 11:31:53","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":213905,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/8cea1a485fe7a63ec0f463de.png"},{"id":86940421,"identity":"d3211c06-9741-4c4e-93cd-fe7ace18c7be","added_by":"auto","created_at":"2025-07-17 11:39:52","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":190962,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/067393630fef55428711189a.png"},{"id":86939612,"identity":"66732518-ad5e-4b3e-ba60-82b68288ff06","added_by":"auto","created_at":"2025-07-17 11:31:52","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":266362,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/940d5d5f363a2660de181860.png"},{"id":86939066,"identity":"6a4458fc-3065-4908-9cc8-d2507756c315","added_by":"auto","created_at":"2025-07-17 11:23:53","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":283049,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/e6bc506172365e78de6bf349.png"},{"id":86939070,"identity":"15a6e48a-00cb-4bfb-aea7-a90b948dffea","added_by":"auto","created_at":"2025-07-17 11:23:53","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":272426,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6954149/v1/b837c42a9f1417b80d7cc2f6.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of the Ecological Safety of Polygenic Cotransformed Populus × euramericana","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eOwing to its fast growth and wide range of uses, poplar is currently being extensively planted worldwide. Nonetheless, as a consequence of the extensive use of only a few poplar clones, a decrease in poplar genetic diversity and serious insect pest-infestation problems have been reported in recent years (Wang et al., 2018). Additionally, owing to the large area with saline-alkali soils in China and the shortage of saline-alkali resistant germplasm, breeding for poplar resistance is an increasingly urgent need. However, the long reproductive cycle and complex reproductive characteristics of this species greatly limit successful breeding of new poplar varieties. Beginning in the 20th century, genetic engineering-assisted plant breeding, has proved effective in accomplishing great directional variety improvement in a short time. Furthermore, it is an effective way to solve the problems of poplar insect infestation and restrictions to a wider geographical distribution (Noushahi and Hussain, 2020).\u003c/p\u003e\n\u003cp\u003eSome elite poplar-hybrid clones have been successively cultivated in China and have entered the stages of intermediate testing, environmental release, production testing, and commercial application (Hu et al., 2010). Insect pest resistance adaptability and non-target insect toxicity in different geographical and climatic environments are key factors for assessing the ecological impact of \u003cem\u003eBt\u003c/em\u003e transgenic plants (Tabashnik et al., 2013; Martinez et al., 2018). Indeed, to date, many countries have already conducted large-scale environmental release tests on \u003cem\u003eBt\u003c/em\u003e transgenic plants, and the first lepidopteran pest resistance to Bt preparation in the world was reported in 1985. Subsequently, a number of laboratory and field studies have confirmed that a range of insects, including Lepidoptera, Coleoptera, Diptera, and nematode species, have developed resistance to the Bt toxin under long-term high-pressure selection (Marroquin and Elyassnia, 2000; Ferré and Van Rie, 2002; Tabashnik and Carrière, 2017; Chandrasena et al., 2018).\u003c/p\u003e\n\u003cp\u003eTabashnik et al. (1990) found resistance in diamondback moths to Bt preparations in field tests conducted in Hawaii, USA, where Bt preparations have been used for a long time. Additionally, after monitoring the susceptibility of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e to \u003cem\u003eCry1F\u003c/em\u003e-modified maize in several regions of Argentina for three years, Chandrasena et al. (2018) were the first to report the detection of \u003cem\u003eS. frugiperda\u003c/em\u003e resistance to the Cry1F toxin in Argentina, and similar results have been obtained in Brazil (Juliano et al., 2014). Further, a study conducted in 2018 in Europe found that field populations of \u003cem\u003eSesamia nonagrioides\u003c/em\u003e increased the frequency of the Cry1Ab resistance allele to 0.0036 after 12 years of cultivation of Cry1Ab-transgenic corn (Camargo et al., 2018). Serine protease inhibitors (serpins) inhibit the conversion of the Cry1Ac proprotein into the activated toxin, resulting in reduced insecticide activity of Cry1Ac against cotton bollworms (Zhang et al., 2021). In turn, the first forced recall of GM varieties owing to resistance issues was reported in Puerto Rico in 2012 (Storer et al., 2012). Furthermore, Fu et al. (2021) determined the virulence of four insecticidal proteins, Cry1Ab, Cry1Ac, Cry1Ah, and Cry1Ca, against the\u0026nbsp;\u003cem\u003eBtk\u003c/em\u003e resistant lines F80 and F100 of cabbage moth and identified the development rules of resistance of cabbage moth-\u003cem\u003eBtk\u003c/em\u003e resistant lines to four Bt insecticidal crystal proteins. The resistance of \u003cem\u003eBtk\u0026nbsp;\u003c/em\u003eresistant lines to Cry1Ac developed rapidly, followed by\u0026nbsp;that to Cry1Ab,\u0026nbsp;whereas resistance to Cry1Ca and Cry1Ah developed more slowly. Further, Fed leaves of transgenic poplar trees carrying the \u003cem\u003eCry3A\u003c/em\u003e gene to plant-eating leaf beetles under greenhouse conditions. Their experiments showed plant resistance to leaf beetles at all stages. (Liliana et al., 2012). Similarly, Yang et al.\u0026nbsp;(2005) conducted a 4-year indoor insect-feeding experiment using 741 poplar trees with double\u0026nbsp;\u003cem\u003eBt\u003c/em\u003e gene transfer and found that the insect resistance effect of the three high-resistance lines was stable, and the larval mortality rate of lepidoptera leaf-eating pests such as \u003cem\u003eClostera anachoreta, Lymantria dispar, Hyphantria cunea\u0026nbsp;\u003c/em\u003ewas higher than 80%, while the development and cocoon formation of surviving larvae were strongly hampered. Consistently, Wang et al. (2014) fed \u003cem\u003eH. cunea\u003c/em\u003e with the leaves of 1-year old poplar seedlings carrying the \u003cem\u003eBt\u003c/em\u003e gene. Although the mortality rates of the different transgenic clones differed, they all showed high insecticidal effects.\u0026nbsp;Additionally, Meilan et al.\u0026nbsp;(2000) conducted field trials on 51\u0026nbsp;\u003cem\u003eCry3A\u003c/em\u003e transgenic poplar trees and found that almost all transgenic lines in eastern Washington showed low insect feeding damage under natural conditions in the wild, whereas non-transgenic lines showed severe leaf damage and an average growth rate that was 13% lower than\u0026nbsp;that of transgenic plants. Furthermore, Chen et al. (2012) investigated the arthropod community of the poplar and cotton complex ecosystem and found that, compared with other complex ecosystems, the transgenic poplar and cotton complex ecosystem had a strong compound inhibitory-effect on the target pests. Altogether, the above studies indicate that the ecological effects of \u003cem\u003eBt\u003c/em\u003e transgenic plants on the ecosystem in the field cannot be generalized.\u003c/p\u003e\n\u003cp\u003eIn the early stages, we linked insect-resistant and saline-alkali-resistant genes into the same vector and introduced them into the poplar genome using the \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation method, which successfully achieved a breakthrough in the breeding of insect-resistant and saline-alkali-resistant poplar cultivars (Liu et al., 2016; Yang et al., 2016). Moreover, transgenic poplars performed well in insect-feeding experiments. In this study, the arthropod community of transgenic polygene 107 poplar (GM 107) was analyzed at different times and in different spaces through a regionalization experiment. The rationale of our study is that it is of the greatest theoretical and practical significance to further study the effects of exogenous gene introduction on the arthropod community, explore the ecological relationship between plants and insects, and control harmful insect populations.\u003c/p\u003e"},{"header":"2 Methodology","content":"\u003cp\u003e2.1 Research material\u003c/p\u003e\n\u003cp\u003eThe transgenic polygene \u003cem\u003ePopulus\u003c/em\u003e × \u003cem\u003eeuramericana\u003c/em\u003e (referred to as GM 107) was cultivated at Hebei Agricultural University. Three lines carrying \u003cem\u003eCry1Ac-Cry3A-BADH\u003c/em\u003e genes (referred to as A1-A3), three lines with \u003cem\u003eCry1Ac-Cry3A-NTHK1\u003c/em\u003e genes (referred to as B1-B3), and the receptor 107 poplar were used as research materials (Liu et al., 2016; Yang et al., 2016). The vector maps of the C\u003cem\u003ery1Ac-Cry3A-BADH\u003c/em\u003e and \u003cem\u003eCry1Ac-Cry3A-NTHK1\u003c/em\u003e genes are shown in Supplementary Figure 1. All six transgenic lines were approved by the National Forestry and Grassland Administration for environmental release planting, and the high-resistance lines were screened as A1 and B1 in\u0026nbsp;the indoor feeding experiments.\u003c/p\u003e\n\u003cp\u003eExperiment design\u003c/p\u003e\n\u003cp\u003eIn April 2018, experimental forests of GM 107 were established in Baoding City, Cangzhou City, and Tangshan City in Hebei Province (Figure 1). A completely randomized blocks design was adopted, and non-GM 107 plants were planted around each block as a border line. The terrain, geomorphology, soil quality, air temperature, vegetation, and cultivation management conditions in the blocks were consistent.\u003c/p\u003e\n\u003cp\u003e2.2 Measuring insect resistance\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInsect resistance was determined at 30-day intervals during the growing season. Six lines were investigated using a random sampling method and five plants were randomly selected from each block of each line. In all east, south, west, and north directions, we recorded the species and number of arthropods in the upper and lower branches, on the trunk within 2 m of the soil surface, and a 1 m×1 m ground surface area around the trunk. For mites and aphids present in large numbers, the number of insects on the third leaf from the tip of the sampled branch was recorded. Most insects were identified to the species level but some only to the family level. According to their feeding characteristics, arthropods in the community were divided into basal, median, top, and into three-nutrient levels (Blondel, 2003). Based on their systematic classification, spatial distribution, and feeding habits, insects in the arthropod communities were divided into different functional groups.\u003c/p\u003e\n\u003cp\u003e2.3 Community feature index\u003c/p\u003e\n\u003cp\u003eTotal number of individuals (\u003cem\u003eN\u003c/em\u003e): the sum of the individuals counted in each tree line.\u003c/p\u003e\n\u003cp\u003eRichness (\u003cem\u003eS\u003c/em\u003e): The total number of arthropod species in the community.\u003c/p\u003e\n\u003cp\u003eRelative abundance: \u003cem\u003eP\u003csub\u003ei\u003c/sub\u003e\u003c/em\u003e=\u003cem\u003eN\u003csub\u003ei\u003c/sub\u003e/N\u003c/em\u003e, where \u003cem\u003eN\u003csub\u003ei\u003c/sub\u003e\u0026nbsp;\u003c/em\u003eis the number of individuals of the \u003cem\u003eith\u003c/em\u003e species and \u003cem\u003eN\u003c/em\u003e is the total number of individuals.\u003c/p\u003e\n\u003cp\u003eDiversity Index (\u003cem\u003eH'\u003c/em\u003e): Shannon-Wiener Diversity index is adopted and the formula is:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"117\" height=\"54\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eCommunity evenness € was calculated using the evenness index proposed by Pielou as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"93\" height=\"19\" src=\"data:image/wmf;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eBerger-Parker dominance index:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC\u0026nbsp;\u003c/em\u003e= \u003cem\u003eN\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e/\u003cem\u003eN\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ewhere,\u0026nbsp;\u003cem\u003eN\u003csub\u003emax\u003c/sub\u003e\u003c/em\u003e is the number of dominant species; \u003cem\u003eN\u003c/em\u003e is the number of individuals of all species.\u003c/p\u003e\n\u003cp\u003e2.4 Community stability\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe ratios of total community species to total community individuals (\u003cem\u003eS\u003csub\u003es\u003c/sub\u003e/S\u003csub\u003et\u003c/sub\u003e\u003c/em\u003e), predatory natural enemies to herbivorous insects (\u003cem\u003eS\u003csub\u003en\u003c/sub\u003e/S\u003csub\u003ep\u003c/sub\u003e\u003c/em\u003e), and\u0026nbsp;predatory natural enemies to herbivorous insects (\u003cem\u003eS\u003csub\u003ea\u003c/sub\u003e/S\u003csub\u003eb\u003c/sub\u003e\u003c/em\u003e) were used to describe the relative stability of the community. While \u003cem\u003eS\u003csub\u003es\u003c/sub\u003e/S\u003csub\u003et\u003c/sub\u003e\u003c/em\u003e reflects the quantitative restrictions among species,\u0026nbsp;\u003cem\u003eS\u003csub\u003en\u003c/sub\u003e/S\u003csub\u003ep\u003c/sub\u003e\u003c/em\u003ereflects the complexity of food web relationships within communities.\u003c/p\u003e\n\u003cp\u003e2.5 Similarity analysis\u003c/p\u003e\n\u003cp\u003eThe total number of arthropods of different orders on different transgenic lines during the whole year was considered as a sample, and the differences in arthropod communities in different groups were tested by non-metric multidimensional scaling (NMDS) based on the Bray-Curtis distance coefficient. After standardization of the original data, the European distance and class average method (WPGMA) were used for clustering.\u003c/p\u003e\n\u003cp\u003e2.6 Primary response curve analysis\u003c/p\u003e\n\u003cp\u003eThe principal response curve (PRC) analysis, is a multivariate analysis of the long-term effects of a treatment. It is suitable for the screening of indicator species, and the statistical results are evaluated by Monte Carlo arrangement tests (Van Wijngaarden et al., 1995).\u003c/p\u003e\n\u003cp\u003e2.7 Statistical analysis\u003c/p\u003e\n\u003cp\u003eExcel was used for statistical analysis of the data. Five surveys were conducted at similar points for inter-year comparative analysis, SPSS was used for variance analysis, \u003cem\u003eR\u003c/em\u003e language was used for PRC analysis, and GraphPad Prism software was used for data visualization. For community analysis purposes, the mean data of lines A1, A2, and A3 were named TA processing, and the mean data of lines B1, B2, and B3 were named TB processing.\u003c/p\u003e"},{"header":"3 Results","content":"\u003cp\u003e3.1 Analysis of exogenous gene stability and expression in GM 107\u003c/p\u003e\n\u003cp\u003eThe PCR amplification products of GM 107 from the test-forest are shown in Supplementary Figure 2. Using \u003cem\u003eCry1Ac\u003c/em\u003e and \u003cem\u003eCry3A\u003c/em\u003e specific primers, 546bp and 667bp bands were amplified from the six transgenic lines and positive plasmids. Additionally, 507bp bands were amplified from the A1, A2, and A3 lines and positive plasmids using\u0026nbsp;\u003cem\u003eBADH\u003c/em\u003e specific primers. In turn, 478bp bands were amplified from\u0026nbsp;the B1, B2, and B3\u0026nbsp;lines and\u0026nbsp;the positive plasmid using\u0026nbsp;\u003cem\u003eNTHK1\u003c/em\u003e specific primers. No bands were amplified in the samples of non-transgenic poplar using the combination of the above primers, indicating that no loss of exogenous genes occurred\u0026nbsp;during the field growth and development of\u0026nbsp;GM 107.\u003c/p\u003e\n\u003cp\u003eAn enzyme-linked immunosorbent assay (ELISA) was used to detect toxins in the highly resistant lines A1 and B1. The results of these assays are summarized in Supplementary Figure 3. Toxic proteins were detected in the leaves, phloem, and roots of A1 and B1lines. The Cry1Ac content was much lower than that of Cry3A, and the difference in Cry1Ac was small in the A1 and B1 lines, whereas the difference in Cry3A was large, and the difference in the B1 line was much higher than that of A1. Additionally, Cry1Ac content was highest in the long and short branches and leaves, followed by the phloem, and lowest in the roots, with significant differences among these organs. Similarly, Cry3A content changes differed in A1 and B1 lines; the expression orders were leaf \u0026gt; phloem \u0026gt; root in the A1 line and phloem \u0026gt; leaf \u0026gt; root in the B1 line.\u003c/p\u003e\n\u003cp\u003e3.2 Arthropod community analysis of GM 107 experimental forest\u003c/p\u003e\n\u003cp\u003eThe survey results of seven lines in the test-forest were used as the sampling times, and 35 samples were collected over three years. The dilution curve showed that the species accumulation curves of several surveys tended to be smooth (Supplementary Figure 4), indicating that the survey results fully reflected the arthropod community at GM 107.\u003c/p\u003e\n\u003cp\u003eFigure 2a-c shows the composition of the arthropod communities at the order level in the Mancheng test-forest for three consecutive years. In 2018, 10485 arthropods belonging to two classes, 10 orders, 48 families, and 71 species were counted; meanwhile, in 2019, 15077 arthropods belonging to three classes, nine orders, 61 families, and 90 species were counted; and in 2020, 8930 arthropods belonging to three classes, 10 orders, 56 families, and 73 species were counted. From the perspective of species richness, the number of species in 2019 and 2020 slightly increased, compared with that in 2018, which may be related to the growth stage of poplars. From the perspective of quantity, Hemiptera, Lepidoptera, and Hymenoptera accounted for a higher proportion, and there were varying degrees of fluctuation over the past three years without an obvious pattern of change.\u003c/p\u003e\n\u003cp\u003eFigure 2d-f shows the composition of arthropod communities at the order level in the experimental forest over different years. In 2018, 5538 arthropods belonging to two classes, nine orders, 49 families, and 64 species were investigated in Yanshan, including 4015 insects and 1523 arachnids. A total of 13098 arthropods belonging to two classes, 10 orders, 50 families, and 68 species were investigated in Luannan, including 12185 insects and 913 spiders, all of which were dominated by lepidopterans.\u003c/p\u003e\n\u003cp\u003eSignificant differences in abundance in the test-forest at the order level in different regions were observed. For example, the relative abundances of Lepidoptera were 33.30% in Mancheng, 36.29% in Yanshan, and 45.83% in Luannan. Meanwhile, the relative abundances of Hemiptera were 18.37% in Mancheng, 5.70% in Yanshan, and 24.91% in Luannan. In turn, the relative abundances of Hymenoptera were 31.66%, 15.20%, and 9.47%, in Mancheng, Yanshan, and Luannan, respectively, indicating that the arthropod community compositions of GM 107 at the different sites were significantly different.\u003c/p\u003e\n\u003cp\u003e3.3 Comparison of arthropod communities in different lines of GM 107 test-forests\u003c/p\u003e\n\u003cp\u003eA Venn diagram shows the species composition of GM 107 (Figure 3a). Overall, transgenic lines and controls exhibited similar species compositions and community structures. In 2018 and 2019, 53 species of control lines were investigated, with the number of genetically modified lines ranging from 49 to 55 and 35 species shared by seven lines. In 2020, 51 species were investigated in the control line and the number of genetically modified lines ranged from 42 to 53, with 28 common species in seven lines. The relative abundance of species fluctuated among lines; however, the overall community structure was similar. Figure 3b shows the quantitative structure of the arthropod communities at the order level. Overall, the number of insect populations on the transgenic lines in the three test stands was lower than that in the control. In addition, although relatively small, differences in the number of insect populations in each transgenic line were observed among them.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 3c, the number of species in each line of Mancheng ranged from 49 to 54, with 32 common species. Additionally, the number of species in line B2 was the highest and those in lines A3 and B3 were the lowest. In turn, the number of species in each line of the Yanshan area ranged from 41 to 50, with 31 common species. In this case, line B2 showed the highest number of species and A1 the lowest. Lastly, the number of species in Luannan ranged from 52 to 67, with 34 common species. In this test-forest, A2 and B2 showed the highest and lowest number of species, respectively. Most arthropod species appeared in both transgenic and control lines, and the number of species fluctuated among lines; however, it was similar between the transgenic and control lines, and there was no significant change. Figure 2d shows the quantitative structure of the arthropod communities at the order level. Overall, the number of insect populations in the transgenic lines in the three test-stands was smaller than that in control stands and differences in the number of insect population among transgenic lines was relatively small.\u003c/p\u003e\n\u003cp\u003e3.4 Community characteristic index of GM 107 lines\u003c/p\u003e\n\u003cp\u003eDiversity reflects community richness and variation. Figure 4a,c,e shows that during 2018-2020, the Shannon diversity index value for each GM 107 line was 2.48-2.68, 2.40-2.64, and 2.60-2.83, respectively. In turn, the Pielou evenness index was 0.69-0.73, 0.67-0.72, and 0.74-0.79, respectively, and the Berger-Parker dominance index was 0.25-0.32, 0.26-0.36, and 0.16-0.28, respectively. These results indicate that, over time, the arthropod diversity and evenness index increased, the dominance index decreased, and community stability increased. Alternatively, the data might suggest that the arthropod community system was not completely established in the early stages of GM 107 establishment, and the arthropod community tended to remain stable as the trees in the test stands grew. A comparison of the community characteristic indices of different lines showed that there was no significant difference between the group of transgenic lines and the control group over three consecutive years.\u003c/p\u003e\n\u003cp\u003eThe Shannon-diversity index values of GM 107 lines in the experimental forests in MC, YS, and LN were 2.48-2.68, 2.63-2.89, and 2.48-2.79, respectively (Figure 4b, d, f). The Pielou evenness index values were 0.69-0.73, 0.77-0.82, and 0.69-0.77, respectively. Finally, the Berger-Parker dominance index values were 0.25-0.32, 0.17-0.24 and 0.18-0.32, respectively. The results of the community characteristic indices of the different sites showed that the community stability at the different sites was as follows: YS \u0026gt; LN \u0026gt; MC. In the test forests in MC and YS there was no significant difference between the group of transgenic lines and the control group. However, the diversity and evenness index values of the transgenic lines in the LN test forest increased whereas dominance decreased, indicating that the transgenic poplar arthropod community in the LN test forest was more stable. The largest number of lepidopterans occurred in the experimental forest in LN, indicating that the transgenic lines at the nodes of the lepidopteran outbreak were beneficial for community stability.\u003c/p\u003e\n\u003cp\u003e3.5 Analysis of insect resistance of GM 107 lines\u003c/p\u003e\n\u003cp\u003eThe occurrence of the target insects is shown in Figure 5. From 2018 to 2020, the number of lepidopteran insects in the MC test forest was stable, and the number of insect populations were 3942, 3380, and 3600, respectively. Populations were mainly composed of Lyonetiidae, Tortricidae, Arctiidae, and Notodontidae, and were dominated by Lyonetiidae. After planting, the number of insects belonging to Tortricidae gradually decreased, and the numbers of insects belonging to Arctiidae and Notodontidae showed an outbreak trend every other year. In turn, the number of insects in the transgenic lines was lower than that in the control line for three consecutive years (a significant difference in 2018), and the number of Lepidopteridae was not significantly different among the transgenic lines. In turn, coleopteran insects showed an increasing trend each year, and the number of insects was 262, 1678 and 2814, respectively, and mainly comprise Chrysomelidae and Coccinellidae. The number of Coleoptera insects on the transgenic lines in 2018 and 2020 was lower than that on the control, and there was no significant difference in 2019.\u003c/p\u003e\n\u003cp\u003eInsects belonging to Lepidopteridae differed significantly among the three sites. Thus, Tortricidae and Phylloscopidae were dominant in the forest in MC. Meanwhile, Phylloscopidae, Tortricidae, and Notodontidae were dominant in the forest in YS, and Arctiidae and Tortricidae were dominant in the forest in LN. The composition of Coleoptera families was similar in the three experimental forests, and they were mainly composed of Chrysomelidae, Coccinellidae, and Carabidae, with Chrysomelidae being dominant. The transgenic lines mainly inhibited insects belonging to Chrysomelidae but had no significant effect on insects belonging to Coccinellidae and Carabidae. The populations of Lepidoptera and Coleoptera in the transgenic lines were significantly smaller than those in the CK in all three test forests.\u003c/p\u003e\n\u003cp\u003ePrincipal response curve (PRC) analysis is applicable to the study of dynamic community changes. Table 1 shows that the first model axis of each survey group was highly representative, and the Monte Carlo test reached significance (except in 2019). These results indicate that the first model axis of the three test-forests was highly representative, and subsequent PRC analysis based on the first model axis was more reliable. With respect to the arthropod community differences between transgenic poplars and the control, seasonal factors accounted for 62.08%, 70.73%, and 61.57%, of the variance respectively, and transgenic events accounted for 14.54%, 5.63%, and 10.68%. Seasonal factors in the forests in YS and LN explained 46.36% and 40.49% of the variance, respectively, and transgene events explained 26.30% and 21.38%, indicating that seasonal factors consistently had a greater effect on poplar arthropod communities than transgene events.\u003c/p\u003e\n\u003cp\u003eFigure 6 shows that, except for 2020, the two transgenic lines showed different degrees of negative deviation. The variation trend of the main response curve in different years differed and the deviation trend of the test-forest in different regions was relatively consistent. In all three experimental forests, the deviations in arthropod communities in transgenic lines were large in August, September, and October 2018. The deviations were most severe in the experimental forest in LN, whereas the differences were greatest in May in 2019 and 2020.\u003c/p\u003e\n\u003cp\u003eThe correlation between species weights and the main response curve in Figure 5 can be interpreted as the closeness (affinity) of each species to the chart. The figure shows species whose absolute values were greater than 0.1. Insects that responded strongly to the PRC curve (species weight \u0026gt; 0.4) in 2018 in the test-forest in MC included Arctiidae, Pyralidae, Lyonetiidae, and Limacodidae. Meanwhile, Thomisidae, Tortricidae, and Formicidae responded strongly to the PRC curve in 2019, whereas Aphididae responded strongly to the PRC curve in 2020 (species weight=1.449). The insects that responded strongly to the PRC curve in the forest in YS included Arctiidae, Chrysomelidae, Notodontidae, and Tortricidae, whereas those in the PRC curve in the forest in LN included Arctiidae, Chrysomelidae, and Notodontidae. Most of these families belong to Lepidoptera or Coleoptera, indicating that the poplar transgenic lines showed obvious anti-insect efficacy in the three test-forests. The weights of the other families were \u0026lt; 0.4. Notably, Coccinellidae responded to the PRC curve for three consecutive years, and the weight values in 2018, 2019, and 2020 were 0.338, 0.256, and 0.173, respectively. Thomisidae responded to the PRC curve across experimental forests in MC, YS, and LN, with weight values of 0.3136, 0.1075, and 0.1313, respectively. The PRC curves of the s in MC and YS simultaneously responded to Cicadellidae, with weights of 0.139 and 0.2156, respectively, while the PRC curves of the forests in MC and LN responded to Phylloxidae, with weights of 0.2309 and 0.2840, respectively.\u003c/p\u003e\n\u003cp\u003e3.6 Arthropod community nutrition-structure of GM 107\u003c/p\u003e\n\u003cp\u003eAccording to the classification of trophic layers and taxa, the nutrient layer richness and taxa composition of arthropods in GM 107 at different sites are shown in Supplementary Figure 5-7. The three test-forests showed the same pattern; that is, the rocky species richness of arthropod groups in TA and TB decreased compared with those in the control lines, whereas the middle and top species richness increased compared with those of the control. In the experimental forest in MC (Supplementary Figure 5), the rocky species richness of the TA and TB arthropod fauna was 0.5652 and 0.5738, respectively, which were 13.19% and 11.87% lower than that of the control (0.6511). In turn, the median species richness was 0.3597 and 0.3523, respectively, which was 20.83% and 18.34% higher than that of the control (0.2977). The top species richness was 0.0747 and 0.0738, which were 46.18% and 44.42 %, higher, respectively, compared with the control (0.0511).\u003c/p\u003e\n\u003cp\u003eIn the forest in YS (Supplementary Figure 6), the species richness of the TA and TB communities was 0.4887 and 0.4922, respectively, i.e., 30.21% and 29.71% lower, respectively, than that of the control (0.7002). In turn, the median species richness was 0.2755 and 0.2667, respectively, which was 82.78% and 88.83% higher than that of the control (0.1459). Lastly, the top species richness was 0.2451 and 0.2318, i.e., 59.26% and 50.62 % higher, respectively, that that of the control (0.1539). Meanwhile, in the test forest in LN (Supplementary Figure 7), the rocky fauna richness of TA and TB arthropods was 0.7733 and 0.5992, respectively, which was 12.82% and 32.45% lower than that of the control group (0.8870). The median species richness was 0.1695 and 0.1143, which was 93.94% and 30.78 % higher, respectively, than that of the control (0.0874). Finally, the top species richness was 0.0572 and 0.0367, i.e., 123.44% and 43.36 %, higher, respectively, than that of the control (0.0256).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe dominant species determines the nature of the community, and when the dominant species change, it may change the structure and nature of the community to a certain extent. The top species were the dominant groups of Tarantula and Thomisids, among which the main species were Chinese tarantulas and Tritaphosa. In turn, the dominant groups of the median species were Heterogyna, Ladybird, Araneidae, Lacewings, and Aphid fly eats, among which the main species were black-brown ants, harlequin ladybirds, turtle ladybirds, and Chinese Caolinids. The composition and dominant groups of the TA and TB lines were basically the same as those of the control base, middle, and top species, and the dominance of the target groups and some other groups changed to some extent as well. For example, the number and dominance of Phytophthira and locust arthropods in TA and TB communities increased in the test forests of MC and YS compared to those in the control group. Lastly, the number of Araneidae in the arthropod communities of the TA and TB lines decreased but their dominance did not decrease significantly.\u003c/p\u003e\n\u003cp\u003eNutrient layer richness and group composition of arthropods in each line of the MC test forest in 2019 and 2020 are shown in Supplementary Figure 8 and Supplementary Figure 9. In 2019 (Supplementary Figure 8), the base species richness of the TA community increased by 2.65% compared with that of the control, whereas that of the TB community decreased by 10.26%, compared with that of the control. The median number of species in the TA community decreased by 3.13% and that in the TB community increased by 7.09%. The top species in TA, increased by 15.78%, and the arthropod community on TB decreased by 4.75%. In terms of trophic groups, the composition and dominant groups on transgenic lines and controls were essentially the same, and the dominance degree and number of some groups showed the same trend. For example, in the base species, the number of lepidopteran insect pests in the communities on TA and TB decreased, and their dominance decreased by 4.59% and 20.48%, respectively, compared with the control. In turn, the number of sawflies and their dominance increased by 171.43% and 100%, respectively, compared to the control. Further, compared to the control group, the number of heteropterans and their dominance increased by 31.62% and 46.15%, respectively, relative to the control. In contrast, the number of locusts and their dominance increased by 114.29% compared to the control. Conversely, the number of Phyllostraca species and their dominance decreased by 32.00% and 36.00%, respectively but the number of leafhoppers and their dominance increased by 136.54% and 65.38%, respectively. Further, the number of median species and their dominance on TA and TB increased by 75.93% and 85.19%, respectively. In particular, the number of dwarf spiders and their dominance increased by 67.21% and 31.15%, respectively; the number of ladybirds and their dominance decreased by 13.48% and 22.38%, respectively. Meanwhile, the number of top species and their dominance on TA and TB lines increased by 129.51% and 70.49%, respectively. In 2020 (Supplementary Figure 9), compared to the control, the basal and top species richness of the transgenic lines decreased, whereas the median species richness increased. Specifically, the base species decreased by 5.91% and 4.48%, top species decreased by 29.73% and 8.11%, and median species increased by 21.36% and 15.95%, respectively. In terms of the abundance and quantity of trophic groups, the number of lepidopterans in TA and TB arthropod communities decreased by 19.54% and 34.82%, those of leafhoppers increased by 75.84% and 63.91%, respectively, and those of Phyllostraca decreased by 25.71% and 31.67%, respectively. The number of Heterogyna species and their dominance increased by 51.70% and 109.22%, respectively. Among the median species, the abundance of lacewings in TA and TB increased by 54.55% and 44.81%, respectively; the abundance of dwarf spiders increased by 67.53% and 83.12%, respectively; and the abundance of thomisids decreased by 45.95% and 8.11%, respectively.\u003c/p\u003e\n\u003cp\u003e3.7 Functional groups and quantitative structure of arthropod community in GM 107 forest\u003c/p\u003e\n\u003cp\u003eThe composition of various groups in the GM 107 forests reflects the complexity of the food network and indicates the extent of pest control. In terms of feeding habits, the abundance of hyphae was the highest and that of parasites was the lowest. There were some differences in the composition and structure of the functional groups between the different years and locations. Compared to the control, the abundance of phytophages decreased, the relative abundance of neutrals increased, and the differences between predatory insects, spiders, and parasites were relatively small (Figure 7a and 7b). In terms of community stability indices, \u003cem\u003eS\u003csub\u003es\u003c/sub\u003e/S\u003csub\u003et\u003c/sub\u003e\u003c/em\u003e, \u003cem\u003eS\u003csub\u003en\u003c/sub\u003e/S\u003csub\u003ep\u003c/sub\u003e\u003c/em\u003e, and \u003cem\u003eS\u003csub\u003ea\u003c/sub\u003e/S\u003csub\u003eb\u003c/sub\u003e\u003c/em\u003e fluctuated greatly among different years and sites in the test forests (Figure 8c and 8d). Particularly, \u003cem\u003eS\u003csub\u003es\u003c/sub\u003e/S\u003csub\u003et\u003c/sub\u003e\u003c/em\u003e of the transgenic lines was higher than that of the control and \u003cem\u003eS\u003csub\u003ea\u003c/sub\u003e/S\u003csub\u003eb\u003c/sub\u003e\u003c/em\u003e was lower than that of the control, indicating that the transgenic lines had a stronger interspecific-restriction effect and higher, natural damage-control efficiency upon arthropod attack.\u003c/p\u003e\n\u003cp\u003e3.8 Arthropod community structure of GM 107\u003c/p\u003e\n\u003cp\u003eNon-metric multidimensional scaling (NMDS) analysis was performed on the arthropod communities on GM 107 lines in different years (Figure 8a). In 2018 and 2019, the arthropod communities in the experimental forest in MC were concentrated in the second and third quadrants, respectively, while in 2020 they were concentrated in the first and fourth quadrants. In the same year, transgenic and control lines could not be distinguished on the first and second axes. The results showed (Figure 9c) that the community was clustered into four groups when λ=0.5. The control and B1 lines, and other lines clustered together in 2018; and 2019 and 2020 were respectively cluster. According to the NMDS of the community structure of the experimental forests in different regions (Figure 8b), the experimental forests in MC and YS were distributed in the third and first quadrants, respectively, whereas the experimental forests in LN were distributed in the third and fourth quadrants. There was a certain degree of deviation between the transgenic and control in all three test stands, which was smaller than the community distance between the test stands. The results of cluster analysis (Figure 9d) were similar to those of the NMDS. This indicated that the arthropod community structure of GM 107 lines was different from that of the control, and the difference was smaller than that among years and planting sites.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe commercialization of genetically modified trees in China is slow, mainly because of ecological safety issues (Valenzuela et al., 2006). The expression of exogenous genes may affect the secretion of nutrients and some secondary metabolites in host plants and play a key role in the feeding of phytophagous insects and natural predators, as well as the host-seeking and egg-laying of insects, thus affecting the arthropod community and even the entire ecosystem functionality and service provision (Schultz, 1988; Turlings and Erb, 2018; Noushahi and Hussain, 2020). Poplar has a long growth cycle, and leaf-eating and stinging insects are directly exposed to the insecticidal protein in \u003cem\u003eBt\u003c/em\u003e transgenic poplar during the entire growth process. Therefore, the impact of transgenic 107 poplar on arthropod communities is key to its ecological safety evaluation. Research has been conducted to assess the safety of genetically modified crops (Ou et al., 2015). Crop insect-resistance plays a major role in insect control, and the results of the assessment of its ecological safety should be a measure of its impact on agricultural ecosystems. Woody plants are often dominant in ecosystems, and their ecological risk assessment is complicated by longevity issues and the difficulty of extrapolating results from small-scale studies to large-scale plantations (Pons et al., 2005; Lu et al., 2010; Dhurua and Gujar, 2011; Bai et al., 2012; Wan et al., 2012; Fabrick et al., 2014). Comprehensive and reliable data can only be obtained through relatively long-term systematic monitoring of genetically modified trees that are released for use in the field.\u003c/p\u003e\n\u003cp\u003eIn previous studies, \u003cem\u003eBt\u003c/em\u003e transgenic poplar trees showed good and stable insect control under field conditions but their toxic effects on different target pests varied (Valenzuela et al., 2022). For example, the toxic effects of \u003cem\u003eCry1Ac\u003c/em\u003e on the four Lepidoptera species were as follows: \u003cem\u003eMicromelalopha troglodyta\u003c/em\u003e \u0026gt; \u003cem\u003eHyphantria cunea\u003c/em\u003e \u0026gt; \u003cem\u003eClostera anachoreta\u003c/em\u003e \u0026gt; \u003cem\u003eLymantria dispar\u003c/em\u003e, and the toxic effects of \u003cem\u003eCry3A\u003c/em\u003e on Coleoptera were as follows: \u003cem\u003ePlagiodera versicolora\u003c/em\u003e \u0026gt; \u003cem\u003eCerambycidae\u003c/em\u003e (Huang et al., 2021). By monitoring target arthropod communities in different regions in the same year and in the same region over three consecutive years, this study found that differences in the sensitivity of different geographical arthropod populations on GM 107 to insecticidal proteins. However, this difference was within the range of natural differences, and the target insect was still sensitive. Additionally, we found that GM 107 shows anti-insect selectivity and that the degree of toxicity to different species of Lepidoptera or Coleoptera varies. With respect to lepidopteran insects with high occurrence rates in the field, their resistance to the tree toxin was as follows: Lamphidae \u0026gt; Canopidae \u0026gt; Borellidae \u0026gt; Leaf stealers \u0026gt; Acanthidae. As for Coleoptera, the effect on Phyllopteridae was strong but weak on ladybirds and beetles. The arthropod community of GM107 was significantly different from that of the control group; however,\u0026nbsp;there were no significant differences among the different transgenic lines. One reason for this may be the small number of target insects occurring\u0026nbsp;during the research. Alternatively,\u0026nbsp;the differences between the field environment and that in the laboratory, and the resistance of\u0026nbsp;the high-resistance transgenic line being lower in the field than indoors may explain the lack of significance of the differences in arthropod communities among transgenic lines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe occurrence of agricultural and forestry insect pests in the field environment is often characterized by suddenness, randomness, diversity, and non-uniformity, and is affected by cultivation mode, climatic conditions (especially spring temperature and rainfall), regional factors and other factors (Fang et al., 1997).Therefore, the effects of transgenic plants on non-target insects and natural enemies are inconsistent. In a study of \u003cem\u003eBt\u003c/em\u003e transgenic crops, most researchers found that Bt toxic proteins have a direct killing effect neither on non-target pests nor on natural enemies, but may have indirect effects on the ecosystem due to changes in intermediate competition or host malnutrition (Catarino et al., 2015). Studies have shown that transferring Cry3A to potatoes has no adverse effects on non-target pests that may come into contact with the crop (Guan et al., 2018). The number of beneficial arthropods in the plots where Bt toxin was sprayed on the leaves was much higher than that in the plots where traditional chemical pesticides were used. In the areas where genetically modified potatoes were grown, aphids were controlled by natural predators alone, whereas aphid populations increased in plots treated with conventional chemical insecticides (non-aphid insecticides). Wang et al. (2003) conducted spot and field surveys of\u0026nbsp;\u003cem\u003eBt\u003c/em\u003e transgenic cotton and conventional cotton fields and found that the larval stock of\u0026nbsp;\u003cem\u003eBt\u003c/em\u003e transgenic cotton was significantly reduced, and the development of the red spider population\u0026nbsp;was faster than that observed in conventional cotton fields\u0026nbsp;(Mendelsohn et al., 2003).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur results differed from those of previous studies. We did not observe any significant difference between species richness of GM 107 and that of the control group. Further, there was no increase in the number of non-target pests or natural enemies, and the relative abundance of natural enemies and neutral insects in the community increased. The relative number of natural enemies reflects community control efficiency. The food sources of natural enemies are not only dependent on pests. Neutral insects are also an important food source for predatory natural enemies, and maintaining a large population of neutral insects plays an important regulatory role in the development of predatory natural enemies, and even the whole arthropod community (Pons et al., 2005). Therefore, GM 107 not only did not increase the number of non-target insects but, additionally, it optimized the composition of the arthropod community and enhanced ecological immunity.\u003c/p\u003e\n\u003cp\u003eChanges in plant traits induced by the introduction of exogenous genes may change a community structure through interspecific relationships, such as food webs. In this study, we systematically evaluated the arthropod communities of two GM 107 types from the perspectives of communities, subcommunities, functional taxa, trophic layers, and functional groups. Although the arthropod community structure of GM 107 differed with growing environment and year, the community structures of the transgenic and control lines were similar. Many characteristics of the community reflected that the community diversity and evenness indices of transgenic lines were higher, whereas the dominance index was lower in some months, and when an outbreak of lepidopteran pests was severe. In terms of the seasonal dynamics of the overall community-characteristic index, the seasonal dynamics of the total community of GM 107 was relatively stable, presumably because of the strong pest resistance of the transgenic poplar forests, where no pest outbreaks occurred. Other community indices also reflected that the arthropod community-nutrition structure of GM 107 was more sustainable than that of the control group; specifically, there were fewer phytophagous insects and neutral arthropods, and the median and apex species increased, showing a beneficial ecological effect.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThe exogenous gene of GM 107 plants was stable in the field, the toxic protein was expressed throughout the growing season, and the expression levels of toxic proteins were significantly different in different Bt types and different tree organs. The changes in the GM 107 population were complex, and the arthropod community characteristic index was relatively stable. When a lepidopteran pest outbreak was severe, the community stability of the transgenic lines was higher than that of the control plants but in the absence of major pest outbreaks in the community, the community stability of the transgenic lines did not significantly differ from that of the control plants. In the field, GM 107 showed stable insecticidal effects against target pests and insect resistance selectivity, but had no inhibitory or proliferative effects on natural enemies or neutral insect populations. GM 107 effectively showed an increased ability for natural pest control and stability of the arthropod community. Furthermore, the arthropod community structure on GM 107 was similar to that on the control trees\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eI\u003c/strong\u003en this study, the field trial of transgenic \u003cem\u003ePopulus × euramericana\u003c/em\u003e '107' was conducted on a limited scale with relatively short monitoring periods. Therefore, further investigations into arthropod communities within these transgenic poplar plantations should involve expanded and continuous monitoring.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEhics, Consent to Participate, and Consent to Publish declarations: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be available on request and were provided within figures of the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u0026quot; in this section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Major Project of Agricultural Biological Breeding (2022ZD0401502), the Provincial Key\u0026nbsp;Research\u0026nbsp;and\u0026nbsp;Development\u0026nbsp;Program\u0026nbsp;of\u0026nbsp;Hebei\u0026nbsp;(21326301D), the Natural Science Foundation of Hebei Province (C2023204098) and the Young Elite Scientists Sponsorship Program by CAST (2023QNRC001)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYali Huang:\u003c/strong\u003e Investigation, Data curation, Performed statistical analysis and visualization, Writing - original draft. \u003cstrong\u003eZijie Zhang\u003c/strong\u003e, \u003cstrong\u003eShijie Wang:\u003c/strong\u003e Statistical analysis and visualization. \u003cstrong\u003eJinMao Wang\u003c/strong\u003e, \u003cstrong\u003eWeizhen Zhang, Chengcheng Li:\u003c/strong\u003e Review the manuscript. \u003cstrong\u003eMinsheng Yang:\u003c/strong\u003e Designed the study, Writing - Review \u0026amp; Editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the District Nursery Field for providing the planting site, as well as the editor and reviewers for valuable suggestions to improve the previous version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp skip=\"true\"\u003eThe English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: https://scientific-publishing.webshop.elsevier.com/\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBai, Y.Y., Yan, R.H., Ye, G.Y., et al. 2012, Field response of aboveground non-target arthropod community to transgenic Bt-Cry1Ab rice plant residues in postharvest seasons. Transgenic Research, 21(5): 1023-1032. doi: 10.1007/s11248-012-9590-6.\u003c/li\u003e\n\u003cli\u003eBlondel, J., 2003. Guilds or functional groups: Does it matter?. 100(2): 223-231.\u003c/li\u003e\n\u003cli\u003eCamargo, A.M., Andow, D.A., Casta\u0026ntilde;era, P., et al. 2018. First detection of a Sesamia nonagrioides resistance allele to Bt maize in Europe. Science Reports, 8(1): 3977. doi:10.1038/s41598-018-21943-4\u003c/li\u003e\n\u003cli\u003eCatarino, R., Ceddia, G., Areal, F.J., et al. 2015. The impact of secondary pests on Bacillus thuringiensis (Bt) crops. Plant Biotechnology Journal, 13(5): 601-612. doi:10.1111/pbi.12363.\u003c/li\u003e\n\u003cli\u003eChandrasena, D.I., Signorini, A.M., Abratti, G., et al. 2018. Characterization of field-evolved resistance to Bacillus thuringiensis-derived Cry1F \u0026delta;-endotoxin in Spodoptera frugiperda populations from Argentina. Pest management science, 74(3): 746-754. doi:10.1002/ps.4776\u003c/li\u003e\n\u003cli\u003eDhurua, S., Gujar, G.T., 2011. Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India. Pest Management Science, 67(8): 898-903.doi: 10.1002/ps.2127\u003c/li\u003e\n\u003cli\u003eFabrick, J.A., Ponnuraj, J., Singh, A., et al. 2014, Alternative splicing and highly variable cadherin transcripts associated with field-evolved resistance of pink bollworm to Bt cotton in India. PloS One, 9(5): e97900. doi: 10.1371/journal.pone.0097900\u003c/li\u003e\n\u003cli\u003eFang, L.L., Zhou, C., Wang R., et al. 2019. RCS Feature-aided insect target tracking algorithm. journal of radars, 8(05):598-605. doi:10.1030.TN.20191011.1730.002\u003c/li\u003e\n\u003cli\u003eFerr\u0026eacute;, J., Van Rie, J., 2002. Biochemistry and genetics of insect resistance to Bacillus thuringiensis. Annual review of entomology, 47: 501-533. doi:10.1146/annurev.ento.47.091201.145234\u003c/li\u003e\n\u003cli\u003eFu W., Tang T., Wang P., et al., 2022, Resistance Development of Four Bt Insecticidal Crystal Proteins against Btk-resistant Strain of Plutella xylostella in the Laboratory. Chinese Journal of Biological Control, 38(02):328-332. doi:10.16409/j.cnki.2095-039x.2021.05.019.\u003c/li\u003e\n\u003cli\u003eGuan, Z.J., Lu, B.S., Huo, Y.L., et al., 2018. Effects of Bt crops on non-target insect pests. Biodiversity Science, 26(06): 636-644. doi:10.17520/biods.2017315\u003c/li\u003e\n\u003cli\u003eHuang, Y., Zhen, Z., Cui, Z., et al. 2021. Growth and arthropod community characteristics of transgenic poplar 741 in an experimental forest. Industrial Crops and Products, 162: 113284. doi:10.1016/j.indcrop.2021.113284\u003c/li\u003e\n\u003cli\u003eHu, J.J., Yang, M.S., Lu, M.Z., 2010. Advances in biosafety studies on transgenic insect-resistant poplars in China. Biodiversity Science. 18(04): 336-345. doi:10.3724/SP.J.1003.2010.336\u003c/li\u003e\n\u003cli\u003eJuliano, R.F., David, A., Andow, b., et al. 2014. Field-evolved resistance to Cry1F maize by Spodoptera frugiperda (Lepidoptera: Noctuidae) in Brazil-ScienceDirect. Crop Protection, 64(3): 150-158. doi:10.1016/j.cropro.2014.06.019\u003c/li\u003e\n\u003cli\u003eLiliana, P.L., Mario, S., Alejandra, B., 2012. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. Fems Microbiology Reviews, 37(1):3-22. doi:10.1111/j.1574-6976.2012.00341.x.\u003c/li\u003e\n\u003cli\u003eLiu, D.Y., Zhang, J., Dong, Y., et al., 2016. Genetic transformation and expression of Cry1Ac\u0026ndash;Cry3A\u0026ndash;NTHK1 genes in Populus \u0026times; euramericana \"Neva\". Acta Physiologiae Plantarum, 2016, 38(7): 1-11. doi:10.1007/s11738-016-2195-6\u003c/li\u003e\n\u003cli\u003eLu, Y., Wu, K., Jiang, Y., et al. 2010, Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China. Science, 328(5982): 1151-1154. doi: 10.1126/science.1187881\u003c/li\u003e\n\u003cli\u003eMartinez, J.C., Caprio, M.A., Friedenberg, N.A., 2018. Density dependence and growth rate: evolutionary effects on resistance development to Bt (\u003cem\u003eBacillus thuringiensis\u003c/em\u003e). Journal of economic entomology. 111(1): 382-390. doi: 10.1093/jee/tox323//\u003c/li\u003e\n\u003cli\u003eMarroquin, L.D., Elyassnia, D., Griffitts, J.S., et al., 2000. Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. Genetics, 155(4): 1693-1699. doi:10.1093/genetics/155.4.1693\u003c/li\u003e\n\u003cli\u003eMeilan, R., Ma, C., Cheng, S., et al. 2000. High levels of Roundup and leaf-beetle resistance in genetically engineered hybrid cottonwoods. Plant Physiology\u003c/li\u003e\n\u003cli\u003eMendelsohn, M., Kough, J., Vaituzis, Z., et al., 2003. Are Bt crops safe?. Nature Biotechnology, 21(9): 003-1009. doi: 10.1038/nbt0903-1003\u003c/li\u003e\n\u003cli\u003eNoushahi, H. A., Hussain, M., 2020. Risk assessment of genetically modified plants: a review. Asian Journal of Research in Crop Science. 1-10. doi:10.9734/ajrcs/2020/v5i330095\u003c/li\u003e\n\u003cli\u003ePons, X., Lumbierres, B., Lopez, C., et al. 2005, Abundance of non-target pests in transgenic Bt-maize: A farm scale study. European Journal of Entomology, 102(1): 73-79\u003c/li\u003e\n\u003cli\u003eStorer, N.P, Kubiszak, M.E., King, J.E., et al., 2012. Status of resistance to Bt maize in Spodoptera frugiperda: Lessons from Puerto Rico. Journal of Invertebrate Pathology. 110(3): 294-300. doi:10.1016/j.jip.2012.04.007\u003c/li\u003e\n\u003cli\u003eTabashnik, B.E., Br\u0026eacute;vault, T., Carri\u0026egrave;re, Y., 2013. Insect resistance to Bet crops: lessons from the first billion acres. Nature biotechnology, 31(6): 510-521. doi:10.1038/nbt.2597\u003c/li\u003e\n\u003cli\u003eTabashnik, B.E., Cushing, N.L., Naomi, F., et al. 1990. Field Development of Resistance to Bacillus thuringiensis in Diamondback Moth (Lepidoptera: Plutellidae). J.econ.entomol, 83(5): 1671-1676. doi:10.1016/0020-7322(90)90018-K\u003c/li\u003e\n\u003cli\u003eTabashnik, B.E., Carri\u0026egrave;re, Y., 2017. Surge in insect resistance to transgenic crops and prospects for sustainability. Nature biotechnology, 35(10): 926-935. doi:10.1038/nbt.3974\u003c/li\u003e\n\u003cli\u003eTurlings, T.C.J., Erb, M., 2018. Tritrophic interactions mediated by herbivore-induced plant volatiles: Mechanisms, ecological relevance, and application potential. Annual Review of Entomology, 63: 433-452. doi:10.1146/annurev-ento-020117-043507\u003c/li\u003e\n\u003cli\u003eValenzuela, S., Balocchi, C., Rodr\u0026iacute;guez, J., 2006. Transgenic trees and forestry biosafety. Electronic Journal of Biotechnology, 9(3): 335-339. doi:10.2225/vol9-issue3-fulltext-22\u003c/li\u003e\n\u003cli\u003eWang, B., Li, H.M., Cao, H.Q., et al. Mechanisms and Applications of Plant-Herbivore-Natural Enemy Tritrophic Interactions Mediated by Volatile Organic Compounds. Scientia Agricultura Sinica, 2021, 54(08): 1653-1672. doi: 10.3864/j.issn.0578-1752.2021.08.007\u003c/li\u003e\n\u003cli\u003eWang, Y.X., Hao, Y.S., Du, J.Z., et al., 2014. Recovery of transgenic Populus plants with modified Bt Cry1Ac gene and their insect-resistance assay. Chinese Agricultural Science Bulletin, 30(28): 23-28.\u003c/li\u003e\n\u003cli\u003eWan, P., Huang, Y., Wu, H., et al. 2012, Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China. PLoS One, 7(1): e29975. doi: 10.1371/journal.pone.0029975\u003c/li\u003e\n\u003cli\u003eYang, M.S., Gao, B.J., Wang, J.M., et al., 2005. Analysis of Main Characteristic of White Poplar Hybrid 741 Transformed Two Insect-Resistant Genes. Scientia silvae sinicae, 2005, 41(01): 91-97.\u003c/li\u003e\n\u003cli\u003eYang, R.L., Wang, A.X., Zhang, J., et al. 2016. Genetic transformation and expression of transgenic lines of Populus \u0026times; euramericana with insect-resistance and salt-tolerance genes. Genetics and Molecular Research : GMR, 15(2): 248-249. doi: 10.4238/gmr.15028635\u003c/li\u003e\n\u003cli\u003eZhang, C., Wei, J., Naing, Z.L., et al. 2021. Endogenous serpin reduces toxicity of Bacillus thuringiensis Cry1Ac against Helicoverpa armigera (H\u0026uuml;bner). Pesticide biochemistry and physiology, 175: 104837. doi:10.1016/j.pestbp.2021.104837\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Transgenic Populus × euramericana, Bacillus thuringiensis genes, Ecological safety, Arthropod","lastPublishedDoi":"10.21203/rs.3.rs-6954149/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6954149/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND:\u003c/strong\u003e Poplar (Populus SPP.) is an important model species in forest research. With the development of transgenic technology, the genetic transformation of poplar with multi-gene resistance to insect pests and saline-alkali stress has been successfully realized. Here, test-forests were established in Baoding City, Cangzhou City, and Tangshan City of Hebei Province to compare the arthropod community of wildtype and transgenic polygene 107 poplar (GM 107) in the field, and thus determine whether the performance of transgenic poplar with field pest resistance and saline-alkali tolerance is ecologically safe.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS:\u003c/strong\u003e The results confirmed the presence of exogenous genes of field-transgenic poplar GM 107 and the stable expression of the corresponding toxic proteins. The arthropod communities of GM 107 varied among different regions in the same year and among different years in the same region. Additionally, GM 107 showed high insect resistance and selectivity. Toxic effects on Lepidoptera were as follows: Micromelalopha troglodyta \u0026gt; Hyphantria cunea \u0026gt; Clostera anachoreta \u0026gt; Lymantria dispar. The toxic effects on Coleoptera were as follows: Plagiodera versicolora \u0026gt; Cerambycidae. Additionally, there was no obvious inhibitory or proliferative effect on natural enemies or neutral insect populations. Arthropods of field-transgenic poplar varied greatly among regions and vintages. Further, upon a severe lepidopteran pest outbreak, the arthropod community of transgenic ecosystems were more stable, and the structures were similar compared to CK.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSION:\u003c/strong\u003e GM 107 effectively showed an increased ability of the arthropod community. Our study provides strong theoretical support for the safe and sustainable application of GM 107.\u003c/p\u003e","manuscriptTitle":"Evaluation of the Ecological Safety of Polygenic Cotransformed Populus × euramericana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 11:23:40","doi":"10.21203/rs.3.rs-6954149/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-30T14:03:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-06T14:11:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50467214342920634846920593633860646437","date":"2025-07-21T10:02:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-20T12:54:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148794593059530067366118216932405925028","date":"2025-07-18T00:25:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332025435210881693965317144613426032301","date":"2025-07-15T16:53:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-15T07:37:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-15T06:54:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-09T09:51:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-07T09:29:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-07-07T09:26:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"915ef16c-d598-4952-8650-dca80df5278b","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:01:20+00:00","versionOfRecord":{"articleIdentity":"rs-6954149","link":"https://doi.org/10.1186/s12870-025-07798-8","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-12-01 15:57:38","publishedOnDateReadable":"December 1st, 2025"},"versionCreatedAt":"2025-07-17 11:23:40","video":"","vorDoi":"10.1186/s12870-025-07798-8","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07798-8","workflowStages":[]},"version":"v1","identity":"rs-6954149","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6954149","identity":"rs-6954149","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}