Effect of Tropic Level and Metamorphosis on the Stable Isotope Discrimination of Ectropis grisescens

Effect of Tropic Level and Metamorphosis on the Stable Isotope Discrimination of Ectropis grisescens | 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 Article Effect of Tropic Level and Metamorphosis on the Stable Isotope Discrimination of Ectropis grisescens Syed Wadood, Xin Li, Hanyi Mei, Chunlin Li, Jing Nie, Wahab Khan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4105359/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Light stable isotopes ( δ 13 C, δ 15 N, δ 2 H, and δ 18 O) of Ectropis grisescens (a leaf-eating pest) were measured at different developmental stages. Isotope values of larval instars, pupae, and adult tissues were determined to understand fractionation patterns at different life stages and to evaluate the tropic shift from food to insect to excrement. The insect’s δ 13 C tissue values were significantly enriched relative to its diet, whereas insect feces were significantly depleted compared to dietary input. Similarly, δ 15 N values of the pest tissue were significantly enriched compared to its diet and this enrichment was most likely due to protein quality since both insufficient protein and a high dietary protein intake have the potential to enrich δ 15 N of bulk body tissues by increasing the protein turnover. The δ 2 H and δ 18 O values also showed significant fractionation compared to diet. The δ 2 H tropic enrichment from plant to larvae and subsequent decrease from larvae to moth is likely due to net enrichment from plant to Ectropis grisescens . Significant correlations between diet, pest tissues and feces were observed for most isotopes. In addition, the metamorphosis of Ectropis grisescens significantly changed the stable isotope ( δ 13 C, δ 15 N, δ 2 H, and δ 18 O) values of the resulting moth. Biological sciences/Ecology Biological sciences/Plant sciences Tropic enrichment Tea plant Metamorphosis Stable isotopes Ectropis grisescens Figures Figure 1 Figure 2 Figure 3 Figure 4 1.0. Introduction Stable isotope analysis using light isotopes ( δ 13 C, δ 15 N, δ 2 H, and δ 18 O) has become one of the most widely employed tools in animal ecology. The study of these isotopes provides insight into how the isotopic composition of dietary resources is incorporated into the tissues of consumers 1 . Isotopic differences between consumer tissues relative to its diet is referred as a discrimination factor and are expressed as ΔX consumer−diet = δ X consumer – δ X diet . Discrimination factors vary among diets, species and tissues within a single species 2 . These differences are associated with variations in macromolecular composition such as amino acids and lipid contents 3 . Tropic enrichment factors for δ 13 C and δ 15 N and the mechanisms that determine the magnitude of discrimination factors have been investigated for various species; however, the incorporation of δ 2 H and δ 18 O values, and their consequent tropic discrimination between dietary resource and consumer tissue have rarely been reported. δ 15 N composition is widely used to assign trophic position of organisms because consumers are enriched in 15 N compared to their diet (up to 3‰), reflecting that 15 N content in animal tissues is biomagnified along the length of a food chain 4 . Conversely, δ 13 C values change is lower (up to 1‰) as carbon moves through the food chain and therefore can be employed to evaluate the basal carbon dietary source for animals when there are multiple dietary carbon sources 5 . δ 13 C is mainly used to differentiate plant-based diets with different photosynthetic pathways such as C 3 , C 4, and CAM in terrestrial and aquatic ecosystems. δ 2 H and δ 18 O isotopes have received an enormous amount of attention from researchers engaged in forensic ecology 6 . The δ 2 H values of consumer tissues can be derived from diet or ingested water. Large differences have been reported in the δ 2 H values between aquatic and terrestrial plants. In an early study, δ 2 H values in aquatic insects and fish were measured and subsequently a mixing model was used to estimate the contribution of terrestrial and aquatic sources to the diets of these animals. It was not clear whether there is fractionation during the synthesis of biomolecules from precursors and body water and it was hypothesized that Δ 2 H tissue−diet = 0 and the contribution of body water hydrogen relative to the hydrogen bound in the organic compounds of tissues is insignificant 7 . Additionally, large δ 2 H variations in the collagen of carnivores and herbivores have been reported, and assumed that these differences were the consequence of a biomagnification effect 8 . Tissue to diet fractionation for δ 13 C, δ 15 N, δ 2 H, and δ 18 O differ largely among different tissues. In case of δ 13 C, lipid content and amino acid composition are the main candidates that impart inter-tissue differences in δ 13 C values. Generally, depletion in 13 C accompanies lipid synthesis, thus some of the variations in δ 13 C is due to tissue lipid content 9 . Similarly, δ 15 N values also differs greatly in tissues. The main difference in δ 15 N values between tissues can be associated with amino acid content. δ 15 N values exhibit variation among the amino acids of primary producers, and this variation seems to be highlighted by the physiological processes of consumers. δ 15 N values of amino acids are known to have a bimodal distribution as some amino acids in animal tissues seem to retain exactly the same δ 15 N composition of food, whereas others showed enrichment (tropic enrichment) due to animal metabolism 2 , 10 . Variations in δ 13 C, δ 15 N, δ 2 H, and δ 18 O in holometabolous insects during metamorphosis have also been reported. Holometabolous insects undergo complex metabolic and physiological processes and additionally, catabolism of existing tissues as well as synthesis of new body tissues, accounting for significant isotopic fractionation during their growth and development 1 , 11 , 12 . Most studies on the incorporation of stable isotope ratios into consumer tissue have primarily reported on animals reliant on multiple dietary sources which makes it difficult to estimate a discrimination factor for some isotopes (i.e. δ 2 H and δ 18 O) because multiple sources of hydrogen (both water and food) are combined to biosynthesize tissues. In contrast, there are no reported studies which have explored isotopic transfer between co-existing tea plant and pest ecosystems. Ectropis grisescens , a destructive leaf-eating pest, exclusively feeds on tea leaves, providing an excellent opportunity to investigate the isotopic fractionation associated with dietary intake, metamorphosis and physiological processes, with little impact from diet. In this context, we investigated δ 13 C, δ 15 N, δ 2 H, and δ 18 O of Ectropis grisescens raised on young and mature tea leaves to investigate the impact of tea leaf differences, tropic level and insect’s metamorphosis on the discrimination of light stable isotopes. 2.0. Material and Methods 2.1 Insects and tea-leaves Young larvae of E. grisescens were raised on fresh Yingshuang cultivar tea leaves under controlled conditions at the Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou. The laboratory conditions were maintained at 23 ± 1°C with 75 to 80% relative humidity and a 10:12 light/dark photoperiod. The larvae were nourished in a 500mL glass bottle sealed with a 9cm plastic culture dish. During feeding, the bottle was inverted for feces collection, with a daily routine of brushing out feces from the culture dish. As the larvae progressed to 4th instar, the bottle was repositioned, and feces were discharged daily. E. grisescens developmental stages from egg to larvae to pupae to adult are shown in Fig. 1 . 2.2 Sample preparation From the second developmental stage, E. grisescens was raised under a controlled dietary regimen involving two distinct tea leaf types characterized by varying tenderness levels: A (tender leaves, first bud and three leaves) and B (mature leaves, fifth leaf) which was maintained until pupation. Tea leaves were collected prior to each instar commencement. Simultaneously, feces collection was carried out daily during the feeding process, and stored at room temperature. Larvae and feces were procured at the end of each instar, while pupae and adult larvae were collected post pupation and emergence, respectively. Rigorous sampling criteria were upheld, with more than 20 larvae collected at the second and third instars, and over 10 larvae at the fourth instars, ensuring two biological replicates per treatment. All the samples were frozen in liquid nitrogen and stored at − 80°C for subsequent analysis. 2.3. Stable isotope ratio analysis Dried, finely powdered tea leaves and E. grisescens (larvae, pupae, adult) along with fecal samples collected at each developmental stage were weighed in duplicate (4.5 to 5.5 mg) and packed into tin capsules (3 mm × 5 mm) for δ 13 C and δ 15 N measurements. Elemental analyzer (Vario Pyro Cube, Elementar, Hanau, Germany) coupled with an isotope ratio mass spectrometer (IsoPrime100, Isoprime Ltd, Manchester, England) was used to analyze the samples. The samples were combusted in a combustion furnace at 1150°C and the reduction of the gasses was carried out at 850°C over copper wire. An inert gas (He) with a flow rate of 230 mL/min was passed through a CentrION prior to mass spectrometry. Acetanilide (Puriss. p.a., Sigma-Aldrich) was used to calibrate elemental %C and %N. Reference standard materials including B2155 (protein, δ 13 C = − 27.0‰, δ 15 N = + 6.0‰), USGS64 (glycine, δ 13 C = − 40.8‰, δ 15 N = + 1.8‰), USGS40 (L-glutamic acid, δ 13 C = − 26.4‰, δ 15 N = − 4.5‰), IAEA-CH-6 (sucrose, δ 13 C = − 10.4‰), and IAEA-N-2 (ammonium sulfate, δ 15 N = + 20.3‰) were used for multipoint calibration. B2155 was supplied by Elemental Microanalysis (United Kingdom). The δ 13 C and δ 15 N values were measured relative to V-PDB and AIR, respectively. For δ 2 H and δ 18 O analysis, samples including diet, pest, feces and reference materials were freeze-dried at − 60°C for 3 days to remove all exchangeable water and subsequently equilibrated in the laboratory exposed to local atmospheric conditions for 5 days prior to H and O analysis. About 1.0 mg powdered sample was packed into silver capsules (6 mm × 4 mm) and analyzed using EA-IRMS. Pyrolysis of samples was achieved at 1450°C to produce gaseous H 2 and CO, respectively. The analytes were transferred into the IRMS for isotope determination. The δ 2 H and δ 18 O values were measured relative to Vienna Standard Mean Ocean Water (V-SMOW). Reference standard materials USGS56 (Mexican ziricote, δ 2 H = − 44.0‰, δ 18 O = + 27.2‰) and USGS54 (Canadian lodgepole pine, δ 2 H = − 150.4‰, δ 18 O = + 17.8‰) were used to calibrate the δ 2 H and δ 18 O measurements. Reference materials were obtained from the International Atomic Energy Agency (IAEA, Austria) and the United States Geological Survey (USGS, United States). The analytical precision for δ 13 C, δ 15 N, δ 2 H and δ 18 O was less than ± 0.1‰, ± 0.2‰, ± 2‰, and 0.5‰, respectively. The delta values ( δ ) were calculated as follows: δ E \(={(R}_{sample}/{R}_{standard})\) − 1 Where δ E represents δ 13 C, δ 15 N, δ 2 H, and δ 18 O whereas R sample and R standard represent the 13 C/ 12 C, 15 N/ 14 N, 2 H/ 1 H or 18 O/ 16 O ratios in samples and references, respectively. 2.3. Statistical analysis Different statistical analyses including one way ANOVA, box and whisker plot, and correlation analysis were applied to the data set. All the analyses were carried out using SPSS version 26 (SPSS Inc., Chicago, USA) and Origin 2022 (Origin Lab Corporation, Northampton, MA, USA). 3.0. Results and Discussion 3.1. Elemental and isotopic contents among diet, pest and feces The %C, %N, δ 13 C, δ 15 N, δ 2 H, and δ 18 O values of pest tissues and feces under two different diet regimens (young and mature tea leaves) were measured and results are shown in Fig. 2 . The isotopic contents among diet, insect and feces were measured during different developmental stages including larvae (4 instars), pupae, and adult. At 1st larval instar, %C values for young tea leaves, insects and feces were 46.5, 44.6, and 47.5%, respectively, followed by 45.8, 44.6, and 46.7% during 2nd instar, 45.9, 46.6, and 47.4 at 3rd instar and 49.8, 49.7 and 47.8% at 4th larval instar, respectively. Post hoc test revealed a significant difference in %C values between diet, insects and feces during different growth stages. The %N values of young tea leaves, insects and feces ranged from 3.4 to 11.42% with the highest values observed for insects and the lowest values found in tea leaves. Post hoc test also showed significant differences in %N of diet, insects and feces. The δ 13 C values ranged from − 29.1 to -26.7‰. The highest δ 13 C values were observed for the pest during different growth stages, followed by tea leaves and the lowest values were found in feces. Duncan’s test showed significant differences among diet, insects and feces. The δ 13 C insect and feces values are directly related to the diet of insect 13 . In general, dietary incorporation of isotopic signals into an organism depends on the metabolic pathways employed during assimilations 14 . The highest δ 13 C difference (~ 1.8‰) between diet and insect body was observed in the first larval instar followed by ~ 1.1‰ in the second larval instar, ~ 1‰ in the third and the lowest difference (~ 0.5‰) was observed in fourth larval instar. A slightly different fractionation pattern was observed for pests fed on older tea leaves (Fig. 2 b). The average fractionation difference between diet and pest was ~ 0.6‰ in the first three larval instars. However, a significant 13 C enrichment (~ 0.4‰) in diet to pest was observed in the fourth larval instar. Mean δ 13 C differences between diet and feces was ~ 0.6‰ for older leaves and ~ 0.7‰ for young tea leaves. The metabolic pathway exhibits a distinct preference for a particular carbon isotope, usually favoring the lighter 12 C, during both anabolism and catabolism that leads to a reduction in 13 C between the substrate and end product. The fractionation into biomass is usually weaker during anabolism than catabolism, and high metabolically active tissues have faster turnover rates as compared to less metabolically active tissues 15 . Several studies report that major biochemical components such as carbohydrates, lipids, and proteins differ in 13 C content and that consumers assimilate components with varying efficiencies that lead to the different carbon isotopic fractionation factors in carnivorous and herbivorous animals 16 . High δ 13 C values observed in Ectropis grisescens fed on young tea leaves is probably due to selective assimilation of amino acids with higher δ 13 C values. Essential amino acids undergo low or no fractionation because they are unaffected by metabolic processes 17 . Insects derive essential amino acids directly from their diet; however, non-essential amino acids combined with carbohydrates and dietary lipids enter the TCA cycle, where carbon from lipids or carbohydrates is incorporated into tissues prior to amino acids, fatty acids or sugar synthesis 18 . It is assumed that higher δ 13 C differences between insects and tea leaves are caused by selective assimilation of non-essential amino acids by Ectropis grisescens that undergo several metabolic reactions involving carbon isotopic fractionation. High amino acid δ 13 C variations causes selective assimilation 18 , leading to a higher δ 13 C discrepancy between diet (tea leaves) and pest. The δ 15 N values showed greater variations among diet, insects and feces. Ectropis grisescens fed on young tea leaves had δ 15 N diet, insect and feces values of 3.8, 7.8, and 4.8‰, respectively during the first larval instar followed by 3.3, 6.7, and 3.1‰ in second larval instar, 3.4, 6.8, and 3.2‰ in third larval instar and 5.3, 7.8, and 3.7% in fourth larval instar. Similar significant statistical differences ( p < 0.05) were observed for Ectropis grisescens raised on older leaves. Mean δ 15 N differences between diet and insects in all four larval instars were 3.4, 2.3, 1.9, and 5.2‰, respectively. The average difference between insect/feces and diet/feces was 3.4‰ and 0.9‰, respectively. The maximum δ 15 N insect value (7.4‰) relative to diet was observed in the adult stage. Lighter 14 N isotopes are preferentially metabolized in many catabolic processes and subsequently excreted in contrast to the heavier isotopes, leading to more positive δ 15 N values retained in the insect’s body tissue relative to its diet 19 . Additionally, enzymatic processes are more rapid for molecules containing lighter isotopes, and therefore lighter isotopes are enriched in animal excretions 20 . Typically δ 15 N values are enriched by 3 to 4‰ in consumers relative to their diet, although large variations among different groups of animals have been reported 21 . In a previous study, high δ 15 N crab tissue values relative to the corresponding diet were reported, and the author assumed that crabs assimilated nutrients from leaf litter despite leaves having a low protein content. However, the assimilated nitrogen doesn’t fulfill the crab’s nitrogen metabolic requirements, leading to the recycling of the body’s internal nitrogen content, which is reflected by a strong increase in tissue δ 15 N values 22 . The same trend was observed in this study for Ectropis grisescens as higher δ 15 N tissue values relative to its diet are not only associated with the insect’s nitrogen assimilation from the tea leaves. Another study reported that a diet lacking in specific dietary nutrients may increase the catabloic rate, ultimately causing additional metabolic cycling of non-essential nutrients that leads to higher δ 15 N values between diet and animal tissue 23 . Both insufficient protein and high protein diets have the potential to enrich δ 15 N of bulk body tissues by increasing the protein turnover. Accordingly, nitrogen content in excretion will rise as the protein intake or catabolism increases, leading to higher δ 15 N values 3 . Other factors, including microbial activity or stress caused by laboratory conditions, may also have contributed to 15 N fractionation. Nitrogen is mainly excreted as urinary urea, where 15 N is remarkably lower than that in the diet consumed. The rate of urinary urea excretion can be increased by stress that can impart significant differences in δ 15 N values between consumer tissues and its diet 24 . In addition, symbiotic bacteria or fungi in the digestive tract of Ectropis grisescens might be involved in nitrogen assimilation that can contribute an additional tropic level in the digestive tract, ultimately reflected as higher δ 15 N values in insect tissue relative to its diet. The δ 2 H values of diet, insects and feces were distinct for both diet regimes. During first larval instar, when Ectropis grisescens were raised on young tea leaves, the highest δ 2 H values were found in feces (-77.5‰), followed by insects (-82.9‰) and the lowest values were found in the host plant leaves (-86.9‰). No significant differences were observed between diet and pest δ 2 H values during the first three larval instars. However, significant differences was observed between diet and pests in the fourth larval instar. Larger δ 2 H differences were observed between diet and pests during the first and third larval instar where δ 2 H values of pests ( Ectropis grisescens ) were significantly enriched (between − 90.3‰ and − 91.9‰) compared to tea leaves (-103.9‰ and − 112.2‰) at ( p < 0.05). A similar δ 2 H fractionation pattern was reported by Peters et al., (2012) for cabbage looper caterpillars ( Trichoplusia ni ) raised on cabbage plants. The assimilation of body water during non-essential amino acid synthesis may be responsible for this hydrogen enrichment 11 , 25 . The δ 2 H values for later insect growth stages (pupae and moth) showed significant deviations from the host plant values. In a similar study, monarch butterfly larvae and adults were raised on milkweed host plants, where butterfly wing keratin showed an insignificant change in δ 2 H values relative to its host plant, reflecting that there was no δ 2 H trophic enrichment 26 . These results were inconsistent with our findings and study difference may be related to tissue-specific tropic effects since the previous study analyzed wing keratin whereas this present study analyzed bulk tissue which contains more lipids and other higher H-content compounds. The δ 2 H tropic enrichment from plant to larvae and subsequent reduction from larvae to moth is likely due to net enrichment between plant and Ectropis grisescens which was consistent with the previous findings for cabbage looper caterpillars 12 . The δ 18 O values also showed significant variations among diet/insect during different life stages. δ 18 O values of younger tea leaves ranged between 16.6‰ to 18.9‰. The δ 18 O values of pest ( Ectropis grisescens ) ranged between 15.7‰ to 18.1‰. The highest values were observed in first larval instar followed by the second (17.7‰), third (15.9‰), and fourth larval instar (15.7‰). The δ 18 O values of feces followed a similar decreasing trend; first larval instar (18.7‰) > second (17.9‰) > third (15.9‰) > fourth (15.3‰), respectively. Older tea leaves showed significant differences between diet/insect in first three larval instars with the highest mean difference between diet and insect found in second larval instar (1.7‰). The δ 18 O values of pupae in both diet regimes (20.6‰ and 21.9‰) were significantly enriched compared to tea leaves 16.6‰ and 17.4‰, respectively. This δ 18 O enrichment phenomenon at the pupal stage remains unknown and requires further exploration but may be related to the rapid growth and fluid swelling of the pupae sac and preferential retention of 18 O. However, all other life stages showed significant depletion of δ 18 O relative to the pest’s diet. Previously, a smiliar trend was reported for chitin δ 18 O values from a tree feeding insect relative to the δ 18 O values of tree cellulose (diet) 27 . Chitin is biosynthesized from glucose molecules which insects ingest from the plant food source. Consequently, insects inherit the evapotranspirative effects from the carbohydrates they consume and differences between the δ 18 O values of pest and diet would result entirely from biochemical fractionation factors 27 . 3.2. Diet, pest tissue and feces correlations Correlations between diet, bulk Ectropis grisescens tissues and their feces were analyzed and results are shown in Fig. 3 . Pearson’s correlation was applied for cases where data showed normal distributions and Spearman’s correlation for those where data was not normally distributed. The significance level was tested at p < 0.05 and 0.01 for all cases. A positive correlation was shown between young leaves and insect tissue (r 2 = 0.41), and feces (r 2 = 0.69), and insect tissue/feces (r 2 = 0.65); however, the correlations were not statistically significant. A significant strong negative correlation was observed between insect tissue and feces (r 2 = -0.76, p < 0.05) with a diet consisting of mature leaves. The δ 13 C values under both diet regimes showed significant correlations for different parameters. Diet/insect tissue showed significant positive correlation (r 2 = 0.20, p < 0.05) for young leaves. A strong significant positive correlation was also observed between insect tissue and fecal samples (r 2 = 0.74, p < 0.05). Similarly, a significant strong positive correlation between insect tissue raised on older tea leaves and feces (r 2 = 0.86, p < 0.01) was recorded. Diet exhibited significant correlation with feces (r 2 = 0.76, p < 0.05) for %N. Other parameters including diet/insect and insect/feces mainly showed an insignificant negative correlation for %N. The δ 15 N values of Ectropis grisescens tissues showed strong significant positive correlations with young tea leaves (r 2 = 0.75, p < 0.05) and feces (r 2 = 0.92, p < 0.05). The δ 2 H and δ 18 O values also exhibited similar significant correlations with young tea leaves. Ectropis grisescens raised on young tea leaves showed significant δ 2 H correlation with diet (r 2 = 0.95, p < 0.01) and feces (r 2 = 0.77, p < 0.01). A young tea leaf diet also showed a significant positive correlation with pest feces (r 2 = 0.79, p < 0.01). In the case of older tea leaves, significant positive correlations were found between diet and pest (r 2 = 0.73, p < 0.01), diet and feces (r 2 = 0.92, p < 0.01) and pest and feces (r 2 = 0.75, p < 0.05). Only δ 18 O value of older tea leaves/feces (r 2 = 0.73, p < 0.05), and pest/feces (r 2 = 0.87, p < 0.01), showed significant positive correlations. 3.3. Isotopic variations of Ectropis grisescens during different developmental stages δ 13 C, δ 15 N, δ 2 H, and δ 18 O values of Ectropis grisescens during their different developmental stages were measured and results are shown in Fig. 4 . Bulk δ 13 C values ranged between − 26.7 to -28.5‰ among different developmental stages and were ranked in the following descending order; second larval instar < first larval instar < third larval instar < fourth larval instar < adult < pupae. The first three larval instars did not show any significant variations; however, a significant δ 13 C depletion ( p < 0.05) was observed as the insect entered in the lateral developmental stages. Similar results were reported for Calliphora vicina blow flies during different developmental stages 17 . It is well established that metabolic shifts during the insect’s developmental stages influence the 13 C isotope fractionation. Like all holometabolous insects, Ectropis grisescens fat body which is mainly composed of lipids (50%) is stored during the larval feeding stages and is responsible for the synthesis, storage, and utilization of biomolecules during insect growth and development. Also fat body contains glycogen which serves as a main source of glucose during the post-feeding larval and pupal stages 28 . Lipids generally exhibit lower δ 13 C values within an organism than proteins and carbohydrates due to the fractionation that occurs during the oxidation of pyruvate to acetyl-CoA 9 . Significant 13 C depletion in the latter growth stages of Ectropis grisescens is likely caused by mobilization of their lipid reserves to synthesize their exoskeleton, reproductive organs, wings, etc. More positive δ 13 C values in early growth stages might be due to the fractionation required for glycogen mobilization during the earlier developmental stages which enriches larvae in 13 C through constant release of 12 C enriched carbon dioxide 17 . The δ 15 N values of different pest developmental stages ranged between 6.7 to 9.4‰ with the highest values found at the adult stage and lowest values at the second larval instar. Post hoc (Duncan’s) test showed significant differences for δ 15 N values among different life stages ( p < 0.05%, Fig. 4 ). Mean 15 N isotopic enrichment from larva to pupa was 0.4‰, and from pupa to adult was 2.1‰. Another study reported significant 15 N enrichment in adult Calliphora vicina blow flies relative to larvae or pupae (2.5‰) 17 . The observed 15 N enrichment is due to fractionation during transamination and deamination of nitrogen-containing compounds throughout the insect’s growth and development phase 17 . Another study reported more positive δ 15 N adult values as compared to δ 15 N larvae for various holometabolous insects including B. mori , G. mellonella , M. sexta , V. cardui , S. haemorrhoidalis 1 . These results suggest that metamorphosis significantly influences the insect’s δ 15 N values between the larval and adult stages. 15 N fractionation variability within holometabolous insects indicates the occurrence of metabolic processes related to the formation of adult tissues and the production of nitrogenous waste during metamorphosis 1 , 29 . δ 2 H values of Ectropis grisescens from larvae to adult stage ranged between − 113.3 to -78.7‰. Continuous depletion in δ 2 H values was observed as insects entered into the advanced developmental stage. Posthoc tests indicate significant differences among different developmental stages ( p < 0.05) and showed the following decreasing trend: pupae < adult < fourth larval instar < third larval instar < first larval instar < second larval instar. In a previous study, compound specific (amino acids) hydrogen isotopes were measured in butterfly tissues where 2 H enriched amino acids were reported for adult tissues relative to earlier life stages. The study hypothesized that holometabolous insects such as B. phileno pupae lose a large percentage of their weight in terms of water during metamorphosis. This substantial loss of water likely leads to 2 H enrichment of the remaining body pool, which is subsequently used to synthesize 2 H enriched amino acids for adult butterfly tissues 25 . Different insect δ 2 H trends were found in our study compared to those of the previous study were mainly because we measured bulk tissue samples and report bulk δ 2 H values, relative to tissue-specific or compound-specific δ 2 H values reported in other studies. Our results are consistent with those reported by Peters et al. for caterpillars (2012). Ectropis grisescens undergo a massive catabolism of existing tissues and synthesize new tissues during pupation using lipid reservoirs. Since lipids are low in 2 H, we assume that a significant 2 H fractionation during the lateral developmental stages is due to deuterium depleted metabolic water produced by lipid catabolism during metamorphosis 30 . The incorporation of hydrogen molecules from body water into the tissues which is synthesized during metamorphosis reasonably explains why adult tissues have significantly lower δ 2 H values than larval tissues. The δ 18 O tissue values of Ectropis grisescens ranged between 15.6 to 20.6‰ at different developmental stages. The highest δ 18 O values were found in pupae (20.6‰) followed by the first larval instar (18.2‰), second larval instar (17.8‰), third larval instar (15.9‰), fourth larval instar (15.7‰), and adult tissues (15.6‰). We found a significant δ 18 O decrease from the first larval instar to fourth instar and then increase for pupae and finally a further decrease from pupae to adult. Our results were inconsistent with previous findings for silkworms, where δ 18 O values decreased from larvae to pupae and then increased again for silkworm cocoon samples 31 . 4.0. Conclusion The δ 13 C, δ 15 N, δ 2 H, and δ 18 O values of young and mature tea leaves, pest tissues and feces were experimentally examined to investigate the tropic enrichment and metamorphosis fractionation for Ectropis grisescens . The δ 13 C values of pests were significantly enriched relative to their diet whereas feces were significantly depleted compared to dietary isotopic values. These fractionation trends can be explained by the fact that metabolic pathways exhibit a distinct preference for the lighter 12 C, during both anabolism and catabolism that leads to a reduction in 13 C between the substrate and end product. Similarly, δ 15 N values of pest tissues were significantly enriched compared to its diet. This enrichment was most likely due to protein quality since insufficient protein intake and access to a diet high in protein both have the potential to enrich 15 N of bulk body tissues by increasing their protein turnover. Different insect life stages were also found to discriminate between tissue and diet δ 15 N values. δ 2 H and δ 18 O values also showed significant fractionation compared to diet. The tropic enrichment of δ 2 H from plant to larvae and subsequent decrease from larvae to moth is likely due to net 2 H enrichment between plant and Ectropis grisescens . Correlation analysis depicted a significant correlation between diet, pest tissues and feces for most isotopes. In addition, metamorphosis of Ectropis grisescens significantly influenced all stable isotopes ( δ 13 C, δ 15 N, δ 2 H, and δ 18 O). Declarations Data Availability The authors declare that the data supporting the findings of this study are available within the paper. Acknowledgement This work was supported by Special Fund of Discipline Construction for Traceability of Agricultural Product (2023-ZAAS). Declaration The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contributions S.A.W designed the experiments and drafted the manuscript, X.L and J.N collected samples and performed the laboratory experiments, H.Y.M and L.C.L performed data analysis, S.Z.S performed isotope analysis, K.M.R and W.A.K review & edited the manuscript, M.J.T and YW.Y conceived the idea, supervised the research, acquired funding, review and edited the manuscript. The final version was approved by all authors. References Tibbets, T. M., Wheeless, L. A. & Del Rio, C. M. 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Measuring Terrestrial Subsidies To Aquatic Food Web Using Stable Isotopes Of Hydrogen. Ecology 88, 1587–1592 (2007). Birchall, J., O’connel, T. C., Heaton, T. H. E. & Hedges, R. E. M. Hydrogen isotope ratios in animal body protein reflect trophic level. J. Anim. Ecol. 74, 877–881 (2005). DeNiro, M. J. & Epstein, S. Mechanism of Carbon Isotope Fractionation Associated with Lipid Synthesis. Science (80-.). 197, 261–263 (1977). Kolasinski, J. et al. Stable isotopes reveal spatial variability in the trophic structure of a macro-benthic invertebrate community in a tropical coral reef. Rapid Commun. Mass Spectrom. 30, 433–446 (2016). Birchall, J., O’connel, T. C., Heaton, T. H. E. & Hedges, R. E. M. Hydrogen isotope ratios in animal body protein reflect trophic level. J. Anim. Ecol. 74, 877–881 (2005). Peters, J. M., Wolf, N., Stricker, C. A., Collier, T. R. & Martínez del Rio, C. Effects of Trophic Level and Metamorphosis on Discrimination of Hydrogen Isotopes in a Plant-Herbivore System. PLoS One 7, e32744 (2012). Pianezze, S. et al. Stable isotope ratio analysis for the characterisation of edible insects. J. Insects as Food Feed 7, 955–964 (2021). Quinby, B. M., Creighton, J. C. & Flaherty, E. A. Stable isotope ecology in insects: a review. Ecol. Entomol. 45, 1231–1246 (2020). Freude, C. & Blaser, M. Carbon Isotope Fractionation during Catabolism and Anabolism in Acetogenic Bacteria Growing on Different Substrates. Appl. Environ. Microbiol. 82, 2728–2737 (2016). Focken, U. & Becker, K. Metabolic fractionation of stable carbon isotopes: implications of different proximate compositions for studies of the aquatic food webs using δ 13 C data. Oecologia 115, 337–343 (1998). Matos, M. P. V., Konstantynova, K. I., Mohr, R. M. & Jackson, G. P. Analysis of the 13C isotope ratios of amino acids in the larvae, pupae and adult stages of Calliphora vicina blow flies and their carrion food sources. Anal. Bioanal. Chem. 410, 7943–7954 (2018). Fantle, M. S., Dittel, A. I., Schwalm, S. M., Epifanio, C. E. & Fogel, M. L. A food web analysis of the juvenile blue crab, Callinectes sapidus, using stable isotopes in whole animals and individual amino acids. Oecologia 120, 416–426 (1999). Feldhaar, H., Gebauer, G. & Blúthgen, N. Stable isotopes: Past and future in exposing secrets of Ant nutrition (hymenoptera: Formicidae). Myrmecological News 13, 3–13 (2009). Peterson, B. J. & Fry, B. Stable Isotopes In Ecosystem Studies. Annu. Rev. Ecol. Syst. 18, 293–320 (1987). Minagawa, M. & Wada, E. Stepwise enrichment of 15N along food chains: Further evidence and the relation between δ15N and animal age. Geochim. Cosmochim. Acta 48, 1135–1140 (1984). Herbon, C. M. & Nordhaus, I. Experimental determination of stable carbon and nitrogen isotope fractionation between mangrove leaves and crabs. Mar. Ecol. Prog. Ser. 490, 91–105 (2013). Martinez del Rio, C. & Wolf, B. O. Mass balance models for animal isotopic ecology. Physiol. consequences Feed. (2004). Ambrose, S. H. Controlled Diet and Climate Experiments on Nitrogen Isotope Ratios of Rats. in Biogeochemical Approaches to Paleodietary Analysis (eds. Ambrose, S. H. & Katzenberg, M. A.) 243–259 (Kluwer Academic Publishers, 2002). doi: 10.1007/0-306-47194-9_12 . Morra, K. E., Newsome, S. D., Graves, G. R. & Fogel, M. L. Physiology Drives Reworking of Amino Acid δ2H and δ13C in Butterfly Tissues. Front. Ecol. Evol. 9, 1–12 (2021). Hobson, K. A., Wassenaar, L. I. & Taylor, O. R. Stable isotopes (δD and δ 13 C) are geographic indicators of natal origins of monarch butterflies in eastern North America. Oecologia 120, 397–404 (1999). Motz, J. Oxygen and hydrogen isotopes in fossil insect chitin as paleoenvironmental indicators. (University of Waterloo, Ontario, Canada, 2000). Arrese, E. L. & Soulages, J. L. Insect Fat Body: Energy, Metabolism, and Regulation. Annu. Rev. Entomol. 55, 207–225 (2010). Vanderklift, M. A. & Ponsard, S. Sources of variation in consumer-diet δ15N enrichment: a meta-analysis. Oecologia 136, 169–182 (2003). Sessions, A. L., Burgoyne, T. W., Schimmelmann, A. & Hayes, J. M. Fractionation of hydrogen isotopes in lipid biosynthesis. Org. Geochem. 30, 1193–1200 (1999). Li, H. et al. A pilot study of stable isotope fractionation in Bombyx mori rearing. Sci. Rep. 13, 6643 (2023). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-4105359","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":284320032,"identity":"8f2da55d-7e40-47fe-9d6f-a8d044734a25","order_by":0,"name":"Syed Wadood","email":"","orcid":"","institution":"aState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products; Institute of Agro-Products Safety and Nutrition, Zhejiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Syed","middleName":"","lastName":"Wadood","suffix":""},{"id":284320033,"identity":"e3c62925-2a09-4bb5-bd76-f49801bef59a","order_by":1,"name":"Xin Li","email":"","orcid":"","institution":"bMinistry of Agriculture Key Laboratory of Tea Quality and Safety Control, Tea Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Li","suffix":""},{"id":284320034,"identity":"d241d497-cd39-4411-919a-058a3fdd78ea","order_by":2,"name":"Hanyi Mei","email":"","orcid":"","institution":"cKey Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture and Rural Affairs of China","correspondingAuthor":false,"prefix":"","firstName":"Hanyi","middleName":"","lastName":"Mei","suffix":""},{"id":284320035,"identity":"9c82cf8c-22ab-4a5e-bb6d-ebde292f422f","order_by":3,"name":"Chunlin Li","email":"","orcid":"","institution":"cKey Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture and Rural Affairs of China","correspondingAuthor":false,"prefix":"","firstName":"Chunlin","middleName":"","lastName":"Li","suffix":""},{"id":284320037,"identity":"339d5849-570f-41f3-b626-e1dc7a243734","order_by":4,"name":"Jing Nie","email":"","orcid":"","institution":"cKey Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture and Rural Affairs of China","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Nie","suffix":""},{"id":284320040,"identity":"8afcc8b2-d855-4b7e-8c3f-ef04d3aedcf3","order_by":5,"name":"Wahab Khan","email":"","orcid":"","institution":"Department of Food Science and technology, University of Home Economics Lahore","correspondingAuthor":false,"prefix":"","firstName":"Wahab","middleName":"","lastName":"Khan","suffix":""},{"id":284320041,"identity":"034e52f9-09ee-49e8-aebf-de182b2b0afd","order_by":6,"name":"Shengzhi Shao","email":"","orcid":"","institution":"aState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products; Institute of Agro-Products Safety and Nutrition, Zhejiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shengzhi","middleName":"","lastName":"Shao","suffix":""},{"id":284320042,"identity":"c0a9ed36-dfd1-449c-93ce-1ac1c46a496b","order_by":7,"name":"Meijun Tang","email":"","orcid":"","institution":"bMinistry of Agriculture Key Laboratory of Tea Quality and Safety Control, Tea Research Institute, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Meijun","middleName":"","lastName":"Tang","suffix":""},{"id":284320043,"identity":"0f98e774-0457-44ae-9e32-f46cea8bb161","order_by":8,"name":"Karyne Rogers","email":"","orcid":"","institution":"National Isotope Centre, GNS Science, 30 Gracefield Road, Lower Hutt 5040, New Zealand","correspondingAuthor":false,"prefix":"","firstName":"Karyne","middleName":"","lastName":"Rogers","suffix":""},{"id":284320044,"identity":"dc986657-39d9-4cce-9db8-a80b87d6f9f6","order_by":9,"name":"Yuwei Yuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYPACGx5+/gbilTMC1abJSM44QJqWwzYGDQlEqje4kWP+4EPNeR4DhgOMHz7mEKfFsHHGsds85swNzJIztxGj5XaOYTMP220ey4YDbMy8RGv58+8cj8GBBFK0MLYdIEGL5P1nhTN7+5J5JGccbCbOL3xnDm/48OObnT0/f/PBDx+J0aJwgMMAygTFDzFAvoH9AXEqR8EoGAWjYOQCAAKBOzjcKDnxAAAAAElFTkSuQmCC","orcid":"","institution":"aState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products; Institute of Agro-Products Safety and Nutrition, Zhejiang Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yuwei","middleName":"","lastName":"Yuan","suffix":""}],"badges":[],"createdAt":"2024-03-15 06:26:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4105359/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4105359/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53775953,"identity":"5ab10115-9b0d-42e2-9b87-a6b1c038c089","added_by":"auto","created_at":"2024-03-30 08:19:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":213574,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopmental stages of \u003cem\u003eEctropis grisescens\u003c/em\u003e: (a) Eggs; (b) Larvae; (c) Pupae; (d) Adult.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4105359/v1/070afe7620666c6eeb5c811f.jpg"},{"id":53775140,"identity":"e6a66c82-8933-4b4e-af1b-39260138f42c","added_by":"auto","created_at":"2024-03-30 08:11:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":153732,"visible":true,"origin":"","legend":"\u003cp\u003eStable isotope bar graphs showing variations among different tea leaves, pest tissues and feces during different developmental stages. (A) insect raised on young leaves. (B) insect raised on older (mature) tea leaves. \u003csup\u003e\u003cstrong\u003ea-d\u003c/strong\u003e\u003c/sup\u003e indicates significant differences (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4105359/v1/c6b9c6489f86d967e1bd85a9.jpg"},{"id":53775137,"identity":"1ba89b12-7e3a-496a-927f-d58814e3d0b8","added_by":"auto","created_at":"2024-03-30 08:11:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":230076,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis between diet, pest tissue and feces. TA (young tea leaves); TB (mature tea leaves); PA (pest raised on young tea leaves); PB (pest raised on mature tea leaves); FA and FB (feces of PA and PB). * indicates significant correlations \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** indicates significant correlations \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4105359/v1/4875527b346e7e9c33d6a595.jpg"},{"id":53775139,"identity":"b72534a7-a962-4064-a3c3-c4f0c66ee52b","added_by":"auto","created_at":"2024-03-30 08:11:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44500,"visible":true,"origin":"","legend":"\u003cp\u003eBox and whisker plots of stable isotopes during different developmental stages. The horizontal line in each box is the median value and the box represents the 25\u003csup\u003eth\u003c/sup\u003e to 75\u003csup\u003eth\u003c/sup\u003e percentile. The whiskers represent the minimum and the maximum non-outlier values. \u003csup\u003e\u003cstrong\u003ea-d\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eletters represent significant differences among life stages.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4105359/v1/c257702e70b9b0d6d97a4ea3.jpg"},{"id":72601878,"identity":"fe7b0d74-3858-41ca-a558-b869e2a111d1","added_by":"auto","created_at":"2024-12-30 09:02:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1289098,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4105359/v1/578260fc-6e3b-4c8e-99a5-229270636fd4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Tropic Level and Metamorphosis on the Stable Isotope Discrimination of Ectropis grisescens","fulltext":[{"header":"1.0. Introduction","content":"\u003cp\u003eStable isotope analysis using light isotopes (\u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO) has become one of the most widely employed tools in animal ecology. The study of these isotopes provides insight into how the isotopic composition of dietary resources is incorporated into the tissues of consumers \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Isotopic differences between consumer tissues relative to its diet is referred as a discrimination factor and are expressed as ΔX\u003csub\u003econsumer\u0026minus;diet\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eδ\u003c/em\u003eX\u003csub\u003econsumer\u003c/sub\u003e \u0026ndash; \u003cem\u003eδ\u003c/em\u003eX\u003csub\u003ediet\u003c/sub\u003e. Discrimination factors vary among diets, species and tissues within a single species \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These differences are associated with variations in macromolecular composition such as amino acids and lipid contents \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Tropic enrichment factors for \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN and the mechanisms that determine the magnitude of discrimination factors have been investigated for various species; however, the incorporation of \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values, and their consequent tropic discrimination between dietary resource and consumer tissue have rarely been reported.\u003c/p\u003e \u003cp\u003e \u003cem\u003eδ\u003c/em\u003e \u003csup\u003e \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e \u003c/sup\u003eN composition is widely used to assign trophic position of organisms because consumers are enriched in \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN compared to their diet (up to 3\u0026permil;), reflecting that \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN content in animal tissues is biomagnified along the length of a food chain \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Conversely, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values change is lower (up to 1\u0026permil;) as carbon moves through the food chain and therefore can be employed to evaluate the basal carbon dietary source for animals when there are multiple dietary carbon sources \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC is mainly used to differentiate plant-based diets with different photosynthetic pathways such as C\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e4,\u003c/sub\u003e and CAM in terrestrial and aquatic ecosystems.\u003c/p\u003e \u003cp\u003e \u003cem\u003eδ\u003c/em\u003e \u003csup\u003e \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e \u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO isotopes have received an enormous amount of attention from researchers engaged in forensic ecology \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values of consumer tissues can be derived from diet or ingested water. Large differences have been reported in the \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values between aquatic and terrestrial plants. In an early study, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values in aquatic insects and fish were measured and subsequently a mixing model was used to estimate the contribution of terrestrial and aquatic sources to the diets of these animals. It was not clear whether there is fractionation during the synthesis of biomolecules from precursors and body water and it was hypothesized that Δ\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003etissue\u0026minus;diet\u003c/sub\u003e = 0 and the contribution of body water hydrogen relative to the hydrogen bound in the organic compounds of tissues is insignificant \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Additionally, large \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH variations in the collagen of carnivores and herbivores have been reported, and assumed that these differences were the consequence of a biomagnification effect \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eTissue to diet fractionation for \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO differ largely among different tissues. In case of \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, lipid content and amino acid composition are the main candidates that impart inter-tissue differences in \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values. Generally, depletion in \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC accompanies lipid synthesis, thus some of the variations in \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC is due to tissue lipid content \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Similarly, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values also differs greatly in tissues. The main difference in \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values between tissues can be associated with amino acid content. \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values exhibit variation among the amino acids of primary producers, and this variation seems to be highlighted by the physiological processes of consumers. \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values of amino acids are known to have a bimodal distribution as some amino acids in animal tissues seem to retain exactly the same \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN composition of food, whereas others showed enrichment (tropic enrichment) due to animal metabolism \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVariations in \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO in holometabolous insects during metamorphosis have also been reported. Holometabolous insects undergo complex metabolic and physiological processes and additionally, catabolism of existing tissues as well as synthesis of new body tissues, accounting for significant isotopic fractionation during their growth and development \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Most studies on the incorporation of stable isotope ratios into consumer tissue have primarily reported on animals reliant on multiple dietary sources which makes it difficult to estimate a discrimination factor for some isotopes (i.e. \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO) because multiple sources of hydrogen (both water and food) are combined to biosynthesize tissues. In contrast, there are no reported studies which have explored isotopic transfer between co-existing tea plant and pest ecosystems.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEctropis grisescens\u003c/em\u003e, a destructive leaf-eating pest, exclusively feeds on tea leaves, providing an excellent opportunity to investigate the isotopic fractionation associated with dietary intake, metamorphosis and physiological processes, with little impact from diet. In this context, we investigated \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO of \u003cem\u003eEctropis grisescens\u003c/em\u003e raised on young and mature tea leaves to investigate the impact of tea leaf differences, tropic level and insect\u0026rsquo;s metamorphosis on the discrimination of light stable isotopes.\u003c/p\u003e"},{"header":"2.0. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Insects and tea-leaves\u003c/h2\u003e \u003cp\u003eYoung larvae of \u003cem\u003eE. grisescens\u003c/em\u003e were raised on fresh Yingshuang cultivar tea leaves under controlled conditions at the Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou. The laboratory conditions were maintained at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with 75 to 80% relative humidity and a 10:12 light/dark photoperiod. The larvae were nourished in a 500mL glass bottle sealed with a 9cm plastic culture dish. During feeding, the bottle was inverted for feces collection, with a daily routine of brushing out feces from the culture dish. As the larvae progressed to 4th instar, the bottle was repositioned, and feces were discharged daily. \u003cem\u003eE. grisescens\u003c/em\u003e developmental stages from egg to larvae to pupae to adult are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sample preparation\u003c/h2\u003e \u003cp\u003eFrom the second developmental stage, \u003cem\u003eE. grisescens\u003c/em\u003e was raised under a controlled dietary regimen involving two distinct tea leaf types characterized by varying tenderness levels: A (tender leaves, first bud and three leaves) and B (mature leaves, fifth leaf) which was maintained until pupation. Tea leaves were collected prior to each instar commencement. Simultaneously, feces collection was carried out daily during the feeding process, and stored at room temperature. Larvae and feces were procured at the end of each instar, while pupae and adult larvae were collected post pupation and emergence, respectively. Rigorous sampling criteria were upheld, with more than 20 larvae collected at the second and third instars, and over 10 larvae at the fourth instars, ensuring two biological replicates per treatment. All the samples were frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Stable isotope ratio analysis\u003c/h2\u003e \u003cp\u003eDried, finely powdered tea leaves and \u003cem\u003eE. grisescens\u003c/em\u003e (larvae, pupae, adult) along with fecal samples collected at each developmental stage were weighed in duplicate (4.5 to 5.5 mg) and packed into tin capsules (3 mm \u0026times; 5 mm) for \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN measurements. Elemental analyzer (Vario Pyro Cube, Elementar, Hanau, Germany) coupled with an isotope ratio mass spectrometer (IsoPrime100, Isoprime Ltd, Manchester, England) was used to analyze the samples. The samples were combusted in a combustion furnace at 1150\u0026deg;C and the reduction of the gasses was carried out at 850\u0026deg;C over copper wire. An inert gas (He) with a flow rate of 230 mL/min was passed through a CentrION prior to mass spectrometry. Acetanilide (Puriss. p.a., Sigma-Aldrich) was used to calibrate elemental %C and %N. Reference standard materials including B2155 (protein, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003eC\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;27.0\u0026permil;, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003eN\u0026thinsp;=\u0026thinsp;+\u0026thinsp;6.0\u0026permil;), USGS64 (glycine, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003eC\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;40.8\u0026permil;, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003eN\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1.8\u0026permil;), USGS40 (L-glutamic acid, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003eC\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;26.4\u0026permil;, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003eN\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;4.5\u0026permil;), IAEA-CH-6 (sucrose, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003eC\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;10.4\u0026permil;), and IAEA-N-2 (ammonium sulfate, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e15\u003c/sup\u003eN\u0026thinsp;=\u0026thinsp;+\u0026thinsp;20.3\u0026permil;) were used for multipoint calibration. B2155 was supplied by Elemental Microanalysis (United Kingdom). The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values were measured relative to V-PDB and AIR, respectively.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO analysis, samples including diet, pest, feces and reference materials were freeze-dried at \u0026minus;\u0026thinsp;60\u0026deg;C for 3 days to remove all exchangeable water and subsequently equilibrated in the laboratory exposed to local atmospheric conditions for 5 days prior to H and O analysis. About 1.0 mg powdered sample was packed into silver capsules (6 mm \u0026times; 4 mm) and analyzed using EA-IRMS. Pyrolysis of samples was achieved at 1450\u0026deg;C to produce gaseous H\u003csub\u003e2\u003c/sub\u003e and CO, respectively. The analytes were transferred into the IRMS for isotope determination. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values were measured relative to Vienna Standard Mean Ocean Water (V-SMOW). Reference standard materials USGS56 (Mexican ziricote, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003eH\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;44.0\u0026permil;, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e18\u003c/sup\u003eO\u0026thinsp;=\u0026thinsp;+\u0026thinsp;27.2\u0026permil;) and USGS54 (Canadian lodgepole pine, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003eH\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;150.4\u0026permil;, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e18\u003c/sup\u003eO\u0026thinsp;=\u0026thinsp;+\u0026thinsp;17.8\u0026permil;) were used to calibrate the \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO measurements. Reference materials were obtained from the International Atomic Energy Agency (IAEA, Austria) and the United States Geological Survey (USGS, United States). The analytical precision for \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO was less than \u0026plusmn;\u0026thinsp;0.1\u0026permil;, \u0026plusmn; 0.2\u0026permil;, \u0026plusmn; 2\u0026permil;, and 0.5\u0026permil;, respectively.\u003c/p\u003e \u003cp\u003eThe delta values (\u003cem\u003eδ\u003c/em\u003e) were calculated as follows:\u003c/p\u003e \u003cp\u003e \u003cem\u003eδ\u003c/em\u003eE \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(={(R}_{sample}/{R}_{standard})\\)\u003c/span\u003e\u003c/span\u003e\u0026minus; 1\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003eδ\u003c/em\u003eE represents \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e N, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO whereas R\u003csub\u003esample\u003c/sub\u003e and R\u003csub\u003estandard\u003c/sub\u003e represent the \u003csup\u003e13\u003c/sup\u003eC/\u003csup\u003e12\u003c/sup\u003eC, \u003csup\u003e15\u003c/sup\u003eN/\u003csup\u003e14\u003c/sup\u003eN, \u003csup\u003e2\u003c/sup\u003eH/\u003csup\u003e1\u003c/sup\u003eH or \u003csup\u003e18\u003c/sup\u003eO/\u003csup\u003e16\u003c/sup\u003eO ratios in samples and references, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Statistical analysis\u003c/h2\u003e \u003cp\u003eDifferent statistical analyses including one way ANOVA, box and whisker plot, and correlation analysis were applied to the data set. All the analyses were carried out using SPSS version 26 (SPSS Inc., Chicago, USA) and Origin 2022 (Origin Lab Corporation, Northampton, MA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3.0. Results and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Elemental and isotopic contents among diet, pest and feces\u003c/h2\u003e \u003cp\u003eThe %C, %N, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values of pest tissues and feces under two different diet regimens (young and mature tea leaves) were measured and results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The isotopic contents among diet, insect and feces were measured during different developmental stages including larvae (4 instars), pupae, and adult. At 1st larval instar, %C values for young tea leaves, insects and feces were 46.5, 44.6, and 47.5%, respectively, followed by 45.8, 44.6, and 46.7% during 2nd instar, 45.9, 46.6, and 47.4 at 3rd instar and 49.8, 49.7 and 47.8% at 4th larval instar, respectively. Post hoc test revealed a significant difference in %C values between diet, insects and feces during different growth stages. The %N values of young tea leaves, insects and feces ranged from 3.4 to 11.42% with the highest values observed for insects and the lowest values found in tea leaves. Post hoc test also showed significant differences in %N of diet, insects and feces.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values ranged from \u0026minus;\u0026thinsp;29.1 to -26.7\u0026permil;. The highest \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values were observed for the pest during different growth stages, followed by tea leaves and the lowest values were found in feces. Duncan\u0026rsquo;s test showed significant differences among diet, insects and feces. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC insect and feces values are directly related to the diet of insect \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In general, dietary incorporation of isotopic signals into an organism depends on the metabolic pathways employed during assimilations \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The highest \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC difference (~\u0026thinsp;1.8\u0026permil;) between diet and insect body was observed in the first larval instar followed by ~\u0026thinsp;1.1\u0026permil; in the second larval instar, ~\u0026thinsp;1\u0026permil; in the third and the lowest difference (~\u0026thinsp;0.5\u0026permil;) was observed in fourth larval instar. A slightly different fractionation pattern was observed for pests fed on older tea leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The average fractionation difference between diet and pest was ~\u0026thinsp;0.6\u0026permil; in the first three larval instars. However, a significant \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC enrichment (~\u0026thinsp;0.4\u0026permil;) in diet to pest was observed in the fourth larval instar. Mean \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC differences between diet and feces was ~\u0026thinsp;0.6\u0026permil; for older leaves and ~\u0026thinsp;0.7\u0026permil; for young tea leaves. The metabolic pathway exhibits a distinct preference for a particular carbon isotope, usually favoring the lighter \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003eC, during both anabolism and catabolism that leads to a reduction in \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC between the substrate and end product. The fractionation into biomass is usually weaker during anabolism than catabolism, and high metabolically active tissues have faster turnover rates as compared to less metabolically active tissues \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeveral studies report that major biochemical components such as carbohydrates, lipids, and proteins differ in \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC content and that consumers assimilate components with varying efficiencies that lead to the different carbon isotopic fractionation factors in carnivorous and herbivorous animals \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. High \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values observed in \u003cem\u003eEctropis grisescens\u003c/em\u003e fed on young tea leaves is probably due to selective assimilation of amino acids with higher \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values. Essential amino acids undergo low or no fractionation because they are unaffected by metabolic processes \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Insects derive essential amino acids directly from their diet; however, non-essential amino acids combined with carbohydrates and dietary lipids enter the TCA cycle, where carbon from lipids or carbohydrates is incorporated into tissues prior to amino acids, fatty acids or sugar synthesis \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. It is assumed that higher \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC differences between insects and tea leaves are caused by selective assimilation of non-essential amino acids by \u003cem\u003eEctropis grisescens\u003c/em\u003e that undergo several metabolic reactions involving carbon isotopic fractionation. High amino acid \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC variations causes selective assimilation \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, leading to a higher \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC discrepancy between diet (tea leaves) and pest.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values showed greater variations among diet, insects and feces. \u003cem\u003eEctropis grisescens\u003c/em\u003e fed on young tea leaves had \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN diet, insect and feces values of 3.8, 7.8, and 4.8\u0026permil;, respectively during the first larval instar followed by 3.3, 6.7, and 3.1\u0026permil; in second larval instar, 3.4, 6.8, and 3.2\u0026permil; in third larval instar and 5.3, 7.8, and 3.7% in fourth larval instar. Similar significant statistical differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were observed for \u003cem\u003eEctropis grisescens\u003c/em\u003e raised on older leaves. Mean \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN differences between diet and insects in all four larval instars were 3.4, 2.3, 1.9, and 5.2\u0026permil;, respectively. The average difference between insect/feces and diet/feces was 3.4\u0026permil; and 0.9\u0026permil;, respectively. The maximum \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN insect value (7.4\u0026permil;) relative to diet was observed in the adult stage. Lighter \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eN isotopes are preferentially metabolized in many catabolic processes and subsequently excreted in contrast to the heavier isotopes, leading to more positive \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values retained in the insect\u0026rsquo;s body tissue relative to its diet \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, enzymatic processes are more rapid for molecules containing lighter isotopes, and therefore lighter isotopes are enriched in animal excretions \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Typically \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values are enriched by 3 to 4\u0026permil; in consumers relative to their diet, although large variations among different groups of animals have been reported \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In a previous study, high \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN crab tissue values relative to the corresponding diet were reported, and the author assumed that crabs assimilated nutrients from leaf litter despite leaves having a low protein content. However, the assimilated nitrogen doesn\u0026rsquo;t fulfill the crab\u0026rsquo;s nitrogen metabolic requirements, leading to the recycling of the body\u0026rsquo;s internal nitrogen content, which is reflected by a strong increase in tissue \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The same trend was observed in this study for \u003cem\u003eEctropis grisescens\u003c/em\u003e as higher \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN tissue values relative to its diet are not only associated with the insect\u0026rsquo;s nitrogen assimilation from the tea leaves. Another study reported that a diet lacking in specific dietary nutrients may increase the catabloic rate, ultimately causing additional metabolic cycling of non-essential nutrients that leads to higher \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values between diet and animal tissue \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Both insufficient protein and high protein diets have the potential to enrich \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN of bulk body tissues by increasing the protein turnover. Accordingly, nitrogen content in excretion will rise as the protein intake or catabolism increases, leading to higher \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOther factors, including microbial activity or stress caused by laboratory conditions, may also have contributed to \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN fractionation. Nitrogen is mainly excreted as urinary urea, where \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN is remarkably lower than that in the diet consumed. The rate of urinary urea excretion can be increased by stress that can impart significant differences in \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values between consumer tissues and its diet \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In addition, symbiotic bacteria or fungi in the digestive tract of \u003cem\u003eEctropis grisescens\u003c/em\u003e might be involved in nitrogen assimilation that can contribute an additional tropic level in the digestive tract, ultimately reflected as higher \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values in insect tissue relative to its diet.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values of diet, insects and feces were distinct for both diet regimes. During first larval instar, when \u003cem\u003eEctropis grisescens\u003c/em\u003e were raised on young tea leaves, the highest \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values were found in feces (-77.5\u0026permil;), followed by insects (-82.9\u0026permil;) and the lowest values were found in the host plant leaves (-86.9\u0026permil;). No significant differences were observed between diet and pest \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values during the first three larval instars. However, significant differences was observed between diet and pests in the fourth larval instar. Larger \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH differences were observed between diet and pests during the first and third larval instar where \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values of pests (\u003cem\u003eEctropis grisescens\u003c/em\u003e) were significantly enriched (between \u0026minus;\u0026thinsp;90.3\u0026permil; and \u0026minus;\u0026thinsp;91.9\u0026permil;) compared to tea leaves (-103.9\u0026permil; and \u0026minus;\u0026thinsp;112.2\u0026permil;) at (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A similar \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH fractionation pattern was reported by Peters et al., (2012) for cabbage looper caterpillars (\u003cem\u003eTrichoplusia ni\u003c/em\u003e) raised on cabbage plants. The assimilation of body water during non-essential amino acid synthesis may be responsible for this hydrogen enrichment \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values for later insect growth stages (pupae and moth) showed significant deviations from the host plant values. In a similar study, monarch butterfly larvae and adults were raised on milkweed host plants, where butterfly wing keratin showed an insignificant change in \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values relative to its host plant, reflecting that there was no \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH trophic enrichment \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. These results were inconsistent with our findings and study difference may be related to tissue-specific tropic effects since the previous study analyzed wing keratin whereas this present study analyzed bulk tissue which contains more lipids and other higher H-content compounds. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH tropic enrichment from plant to larvae and subsequent reduction from larvae to moth is likely due to net enrichment between plant and \u003cem\u003eEctropis grisescens\u003c/em\u003e which was consistent with the previous findings for cabbage looper caterpillars \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values also showed significant variations among diet/insect during different life stages. \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values of younger tea leaves ranged between 16.6\u0026permil; to 18.9\u0026permil;. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values of pest (\u003cem\u003eEctropis grisescens\u003c/em\u003e) ranged between 15.7\u0026permil; to 18.1\u0026permil;. The highest values were observed in first larval instar followed by the second (17.7\u0026permil;), third (15.9\u0026permil;), and fourth larval instar (15.7\u0026permil;). The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values of feces followed a similar decreasing trend; first larval instar (18.7\u0026permil;)\u0026thinsp;\u0026gt;\u0026thinsp;second (17.9\u0026permil;)\u0026thinsp;\u0026gt;\u0026thinsp;third (15.9\u0026permil;)\u0026thinsp;\u0026gt;\u0026thinsp;fourth (15.3\u0026permil;), respectively. Older tea leaves showed significant differences between diet/insect in first three larval instars with the highest mean difference between diet and insect found in second larval instar (1.7\u0026permil;). The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values of pupae in both diet regimes (20.6\u0026permil; and 21.9\u0026permil;) were significantly enriched compared to tea leaves 16.6\u0026permil; and 17.4\u0026permil;, respectively. This \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO enrichment phenomenon at the pupal stage remains unknown and requires further exploration but may be related to the rapid growth and fluid swelling of the pupae sac and preferential retention of \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO. However, all other life stages showed significant depletion of \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO relative to the pest\u0026rsquo;s diet. Previously, a smiliar trend was reported for chitin \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values from a tree feeding insect relative to the \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values of tree cellulose (diet) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Chitin is biosynthesized from glucose molecules which insects ingest from the plant food source. Consequently, insects inherit the evapotranspirative effects from the carbohydrates they consume and differences between the \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values of pest and diet would result entirely from biochemical fractionation factors \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Diet, pest tissue and feces correlations\u003c/h2\u003e \u003cp\u003eCorrelations between diet, bulk \u003cem\u003eEctropis grisescens\u003c/em\u003e tissues and their feces were analyzed and results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Pearson\u0026rsquo;s correlation was applied for cases where data showed normal distributions and Spearman\u0026rsquo;s correlation for those where data was not normally distributed. The significance level was tested at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and 0.01 for all cases. A positive correlation was shown between young leaves and insect tissue (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.41), and feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.69), and insect tissue/feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.65); however, the correlations were not statistically significant. A significant strong negative correlation was observed between insect tissue and feces (r\u003csup\u003e2\u003c/sup\u003e = -0.76, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with a diet consisting of mature leaves. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values under both diet regimes showed significant correlations for different parameters. Diet/insect tissue showed significant positive correlation (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.20, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for young leaves. A strong significant positive correlation was also observed between insect tissue and fecal samples (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.74, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, a significant strong positive correlation between insect tissue raised on older tea leaves and feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.86, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was recorded.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDiet exhibited significant correlation with feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.76, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for %N. Other parameters including diet/insect and insect/feces mainly showed an insignificant negative correlation for %N. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values of \u003cem\u003eEctropis grisescens\u003c/em\u003e tissues showed strong significant positive correlations with young tea leaves (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.75, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.92, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values also exhibited similar significant correlations with young tea leaves. \u003cem\u003eEctropis grisescens\u003c/em\u003e raised on young tea leaves showed significant \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH correlation with diet (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.95, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.77, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A young tea leaf diet also showed a significant positive correlation with pest feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.79, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the case of older tea leaves, significant positive correlations were found between diet and pest (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.73, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), diet and feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.92, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and pest and feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.75, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Only \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO value of older tea leaves/feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.73, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and pest/feces (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.87, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), showed significant positive correlations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Isotopic variations of \u003cem\u003eEctropis grisescens\u003c/em\u003e during different developmental stages\u003c/h2\u003e \u003cp\u003e \u003cem\u003eδ\u003c/em\u003e \u003csup\u003e \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e \u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values of \u003cem\u003eEctropis grisescens\u003c/em\u003e during their different developmental stages were measured and results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Bulk \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values ranged between \u0026minus;\u0026thinsp;26.7 to -28.5\u0026permil; among different developmental stages and were ranked in the following descending order; second larval instar\u0026thinsp;\u0026lt;\u0026thinsp;first larval instar\u0026thinsp;\u0026lt;\u0026thinsp;third larval instar\u0026thinsp;\u0026lt;\u0026thinsp;fourth larval instar\u0026thinsp;\u0026lt;\u0026thinsp;adult\u0026thinsp;\u0026lt;\u0026thinsp;pupae. The first three larval instars did not show any significant variations; however, a significant \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC depletion (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed as the insect entered in the lateral developmental stages. Similar results were reported for \u003cem\u003eCalliphora vicina\u003c/em\u003e blow flies during different developmental stages \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. It is well established that metabolic shifts during the insect\u0026rsquo;s developmental stages influence the \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC isotope fractionation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLike all holometabolous insects, \u003cem\u003eEctropis grisescens\u003c/em\u003e fat body which is mainly composed of lipids (50%) is stored during the larval feeding stages and is responsible for the synthesis, storage, and utilization of biomolecules during insect growth and development. Also fat body contains glycogen which serves as a main source of glucose during the post-feeding larval and pupal stages \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Lipids generally exhibit lower \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values within an organism than proteins and carbohydrates due to the fractionation that occurs during the oxidation of pyruvate to acetyl-CoA \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Significant \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC depletion in the latter growth stages of \u003cem\u003eEctropis grisescens\u003c/em\u003e is likely caused by mobilization of their lipid reserves to synthesize their exoskeleton, reproductive organs, wings, etc. More positive \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values in early growth stages might be due to the fractionation required for glycogen mobilization during the earlier developmental stages which enriches larvae in \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC through constant release of \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003eC enriched carbon dioxide \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values of different pest developmental stages ranged between 6.7 to 9.4\u0026permil; with the highest values found at the adult stage and lowest values at the second larval instar. Post hoc (Duncan\u0026rsquo;s) test showed significant differences for \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values among different life stages (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05%, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Mean \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN isotopic enrichment from larva to pupa was 0.4\u0026permil;, and from pupa to adult was 2.1\u0026permil;. Another study reported significant \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN enrichment in adult \u003cem\u003eCalliphora vicina\u003c/em\u003e blow flies relative to larvae or pupae (2.5\u0026permil;) \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The observed \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN enrichment is due to fractionation during transamination and deamination of nitrogen-containing compounds throughout the insect\u0026rsquo;s growth and development phase \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Another study reported more positive \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN\u003csub\u003eadult\u003c/sub\u003e values as compared to \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN\u003csub\u003elarvae\u003c/sub\u003e for various holometabolous insects including \u003cem\u003eB. mori\u003c/em\u003e, \u003cem\u003eG. mellonella\u003c/em\u003e, \u003cem\u003eM. sexta\u003c/em\u003e, \u003cem\u003eV. cardui\u003c/em\u003e, \u003cem\u003eS. haemorrhoidalis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. These results suggest that metamorphosis significantly influences the insect\u0026rsquo;s \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values between the larval and adult stages. \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN fractionation variability within holometabolous insects indicates the occurrence of metabolic processes related to the formation of adult tissues and the production of nitrogenous waste during metamorphosis \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eδ\u003c/em\u003e \u003csup\u003e \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e \u003c/sup\u003eH values of \u003cem\u003eEctropis grisescens\u003c/em\u003e from larvae to adult stage ranged between \u0026minus;\u0026thinsp;113.3 to -78.7\u0026permil;. Continuous depletion in \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values was observed as insects entered into the advanced developmental stage. Posthoc tests indicate significant differences among different developmental stages (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and showed the following decreasing trend: pupae\u0026thinsp;\u0026lt;\u0026thinsp;adult\u0026thinsp;\u0026lt;\u0026thinsp;fourth larval instar\u0026thinsp;\u0026lt;\u0026thinsp;third larval instar\u0026thinsp;\u0026lt;\u0026thinsp;first larval instar\u0026thinsp;\u0026lt;\u0026thinsp;second larval instar. In a previous study, compound specific (amino acids) hydrogen isotopes were measured in butterfly tissues where \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH enriched amino acids were reported for adult tissues relative to earlier life stages. The study hypothesized that holometabolous insects such as \u003cem\u003eB. phileno\u003c/em\u003e pupae lose a large percentage of their weight in terms of water during metamorphosis. This substantial loss of water likely leads to \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH enrichment of the remaining body pool, which is subsequently used to synthesize \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH enriched amino acids for adult butterfly tissues \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Different insect \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH trends were found in our study compared to those of the previous study were mainly because we measured bulk tissue samples and report bulk \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values, relative to tissue-specific or compound-specific \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values reported in other studies. Our results are consistent with those reported by Peters et al. for caterpillars (2012). \u003cem\u003eEctropis grisescens\u003c/em\u003e undergo a massive catabolism of existing tissues and synthesize new tissues during pupation using lipid reservoirs. Since lipids are low in \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, we assume that a significant \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH fractionation during the lateral developmental stages is due to deuterium depleted metabolic water produced by lipid catabolism during metamorphosis \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The incorporation of hydrogen molecules from body water into the tissues which is synthesized during metamorphosis reasonably explains why adult tissues have significantly lower \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH values than larval tissues.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO tissue values of \u003cem\u003eEctropis grisescens\u003c/em\u003e ranged between 15.6 to 20.6\u0026permil; at different developmental stages. The highest \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values were found in pupae (20.6\u0026permil;) followed by the first larval instar (18.2\u0026permil;), second larval instar (17.8\u0026permil;), third larval instar (15.9\u0026permil;), fourth larval instar (15.7\u0026permil;), and adult tissues (15.6\u0026permil;). We found a significant \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO decrease from the first larval instar to fourth instar and then increase for pupae and finally a further decrease from pupae to adult. Our results were inconsistent with previous findings for silkworms, where \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values decreased from larvae to pupae and then increased again for silkworm cocoon samples \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"4.0. Conclusion","content":"\u003cp\u003eThe \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values of young and mature tea leaves, pest tissues and feces were experimentally examined to investigate the tropic enrichment and metamorphosis fractionation for \u003cem\u003eEctropis grisescens\u003c/em\u003e. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC values of pests were significantly enriched relative to their diet whereas feces were significantly depleted compared to dietary isotopic values. These fractionation trends can be explained by the fact that metabolic pathways exhibit a distinct preference for the lighter \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003eC, during both anabolism and catabolism that leads to a reduction in \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC between the substrate and end product. Similarly, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values of pest tissues were significantly enriched compared to its diet. This enrichment was most likely due to protein quality since insufficient protein intake and access to a diet high in protein both have the potential to enrich \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN of bulk body tissues by increasing their protein turnover. Different insect life stages were also found to discriminate between tissue and diet \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values. \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values also showed significant fractionation compared to diet. The tropic enrichment of \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH from plant to larvae and subsequent decrease from larvae to moth is likely due to net \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH enrichment between plant and \u003cem\u003eEctropis grisescens\u003c/em\u003e. Correlation analysis depicted a significant correlation between diet, pest tissues and feces for most isotopes. In addition, metamorphosis of \u003cem\u003eEctropis grisescens\u003c/em\u003e significantly influenced all stable isotopes (\u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Special Fund of Discipline Construction for Traceability of Agricultural Product (2023-ZAAS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.A.W designed the experiments and drafted the manuscript, X.L and J.N collected samples and performed the laboratory experiments, H.Y.M and L.C.L performed data analysis, S.Z.S performed isotope analysis, K.M.R and W.A.K review \u0026amp; edited the manuscript, M.J.T and YW.Y conceived the idea, supervised the research, acquired funding, review and edited the manuscript. The final version was approved by all authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTibbets, T. M., Wheeless, L. A. \u0026amp; Del Rio, C. M. Isotopic enrichment without change in diet: an ontogenetic shift in δ 15 N during insect metamorphosis. Funct. Ecol. 22, 109\u0026ndash;113 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolf, N., Carleton, S. A. \u0026amp; Mart\u0026iacute;nez del Rio, C. Ten years of experimental animal isotopic ecology. Funct. Ecol. 23, 17\u0026ndash;26 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlorin, S. T., Felicetti, L. A. \u0026amp; Robbins, C. T. The biological basis for understanding and predicting dietary-induced variation in nitrogen and sulphur isotope ratio discrimination. Funct. Ecol. 25, 519\u0026ndash;526 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePost, D. M. 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E. \u0026amp; Hedges, R. E. M. Hydrogen isotope ratios in animal body protein reflect trophic level. J. Anim. Ecol. 74, 877\u0026ndash;881 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeNiro, M. J. \u0026amp; Epstein, S. Mechanism of Carbon Isotope Fractionation Associated with Lipid Synthesis. Science (80-.). 197, 261\u0026ndash;263 (1977).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKolasinski, J. \u003cem\u003eet al.\u003c/em\u003e Stable isotopes reveal spatial variability in the trophic structure of a macro-benthic invertebrate community in a tropical coral reef. Rapid Commun. Mass Spectrom. 30, 433\u0026ndash;446 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirchall, J., O\u0026rsquo;connel, T. C., Heaton, T. H. E. \u0026amp; Hedges, R. E. M. Hydrogen isotope ratios in animal body protein reflect trophic level. J. Anim. Ecol. 74, 877\u0026ndash;881 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeters, J. M., Wolf, N., Stricker, C. A., Collier, T. R. \u0026amp; Mart\u0026iacute;nez del Rio, C. Effects of Trophic Level and Metamorphosis on Discrimination of Hydrogen Isotopes in a Plant-Herbivore System. PLoS One 7, e32744 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePianezze, S. \u003cem\u003eet al.\u003c/em\u003e Stable isotope ratio analysis for the characterisation of edible insects. J. Insects as Food Feed 7, 955\u0026ndash;964 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuinby, B. M., Creighton, J. C. \u0026amp; Flaherty, E. A. Stable isotope ecology in insects: a review. Ecol. Entomol. 45, 1231\u0026ndash;1246 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreude, C. \u0026amp; Blaser, M. Carbon Isotope Fractionation during Catabolism and Anabolism in Acetogenic Bacteria Growing on Different Substrates. Appl. Environ. Microbiol. 82, 2728\u0026ndash;2737 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFocken, U. \u0026amp; Becker, K. Metabolic fractionation of stable carbon isotopes: implications of different proximate compositions for studies of the aquatic food webs using δ 13 C data. Oecologia 115, 337\u0026ndash;343 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatos, M. P. V., Konstantynova, K. I., Mohr, R. M. \u0026amp; Jackson, G. P. Analysis of the 13C isotope ratios of amino acids in the larvae, pupae and adult stages of Calliphora vicina blow flies and their carrion food sources. Anal. Bioanal. Chem. 410, 7943\u0026ndash;7954 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFantle, M. S., Dittel, A. I., Schwalm, S. M., Epifanio, C. E. \u0026amp; Fogel, M. L. 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Experimental determination of stable carbon and nitrogen isotope fractionation between mangrove leaves and crabs. Mar. Ecol. Prog. Ser. 490, 91\u0026ndash;105 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez del Rio, C. \u0026amp; Wolf, B. O. Mass balance models for animal isotopic ecology. Physiol. consequences Feed. (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmbrose, S. H. Controlled Diet and Climate Experiments on Nitrogen Isotope Ratios of Rats. in \u003cem\u003eBiogeochemical Approaches to Paleodietary Analysis\u003c/em\u003e (eds. Ambrose, S. H. \u0026amp; Katzenberg, M. A.) 243\u0026ndash;259 (Kluwer Academic Publishers, 2002). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/0-306-47194-9_12\u003c/span\u003e\u003cspan address=\"10.1007/0-306-47194-9_12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorra, K. E., Newsome, S. D., Graves, G. R. \u0026amp; Fogel, M. L. Physiology Drives Reworking of Amino Acid δ2H and δ13C in Butterfly Tissues. Front. Ecol. Evol. 9, 1\u0026ndash;12 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHobson, K. A., Wassenaar, L. I. \u0026amp; Taylor, O. R. Stable isotopes (δD and δ 13 C) are geographic indicators of natal origins of monarch butterflies in eastern North America. Oecologia 120, 397\u0026ndash;404 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMotz, J. Oxygen and hydrogen isotopes in fossil insect chitin as paleoenvironmental indicators. (University of Waterloo, Ontario, Canada, 2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArrese, E. L. \u0026amp; Soulages, J. L. Insect Fat Body: Energy, Metabolism, and Regulation. Annu. Rev. Entomol. 55, 207\u0026ndash;225 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVanderklift, M. A. \u0026amp; Ponsard, S. Sources of variation in consumer-diet δ15N enrichment: a meta-analysis. Oecologia 136, 169\u0026ndash;182 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSessions, A. L., Burgoyne, T. W., Schimmelmann, A. \u0026amp; Hayes, J. M. Fractionation of hydrogen isotopes in lipid biosynthesis. Org. Geochem. 30, 1193\u0026ndash;1200 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, H. \u003cem\u003eet al.\u003c/em\u003e A pilot study of stable isotope fractionation in Bombyx mori rearing. Sci. Rep. 13, 6643 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tropic enrichment, Tea plant, Metamorphosis, Stable isotopes, Ectropis grisescens","lastPublishedDoi":"10.21203/rs.3.rs-4105359/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4105359/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLight stable isotopes (\u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO) of \u003cem\u003eEctropis grisescens\u003c/em\u003e (a leaf-eating pest) were measured at different developmental stages. Isotope values of larval instars, pupae, and adult tissues were determined to understand fractionation patterns at different life stages and to evaluate the tropic shift from food to insect to excrement. The insect\u0026rsquo;s \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC tissue values were significantly enriched relative to its diet, whereas insect feces were significantly depleted compared to dietary input. Similarly, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN values of the pest tissue were significantly enriched compared to its diet and this enrichment was most likely due to protein quality since both insufficient protein and a high dietary protein intake have the potential to enrich \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN of bulk body tissues by increasing the protein turnover. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO values also showed significant fractionation compared to diet. The \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH tropic enrichment from plant to larvae and subsequent decrease from larvae to moth is likely due to net enrichment from plant to \u003cem\u003eEctropis grisescens\u003c/em\u003e. Significant correlations between diet, pest tissues and feces were observed for most isotopes. In addition, the metamorphosis of \u003cem\u003eEctropis grisescens\u003c/em\u003e significantly changed the stable isotope (\u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003eN, \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eH, and \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003eO) values of the resulting moth.\u003c/p\u003e","manuscriptTitle":"Effect of Tropic Level and Metamorphosis on the Stable Isotope Discrimination of Ectropis grisescens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-30 08:10:59","doi":"10.21203/rs.3.rs-4105359/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f6a61f4c-0a38-4c60-8420-cce4bd21ee56","owner":[],"postedDate":"March 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29930314,"name":"Biological sciences/Ecology"},{"id":29930315,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2024-12-30T08:54:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-30 08:10:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4105359","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4105359","identity":"rs-4105359","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}