Subchronic Neurotoxic Effects of Cypermethrin on Earthworms

Subchronic Neurotoxic Effects of Cypermethrin on Earthworms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Subchronic Neurotoxic Effects of Cypermethrin on Earthworms Fuhui Zhao, Sijia Wu, Shiping Zhou, Huijuan Li, Qisheng Li, ShouQing Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4097539/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 Cypermethrin is one of the most heavily used pyrethroid pesticides worldwide and is a potential threat to soil organisms such as earthworms. In this paper, Amynthas corticis was selected as a test organism to investigate the neurobehavioral changes in movement, cognition and memory caused by subchronic neurotoxicity of cypermethrin in earthworms, starting from the changes in the characteristic enzymes of earthworms' nerve ion channels triggered by exposure to cypermethrin. The changes in biomarkers of earthworms were evaluated using the integrated biomarker response (IBR), and the mechanism of cypermethrin neurotoxicity in earthworms was investigated using molecular docking technology, so as to investigate the subchronic neurotoxicity of earthworms caused by exposure to cypermethrin. The results showed that the subchronic neurotoxicity of cypermethrin for earthworms increased with the increase of the exposure concentration and the duration of cypermethrin exposure. The chronic neurotoxicity of cypermethrin did not lead to earthworm death but induced neurobehavioral changes such as locomotor retardation and cognitive deficits in earthworms. Cypermethrin exposure induced abnormalities in the enzyme that characterizes nerve ion channels in earthworms, which is one of the possible molecular mechanisms for the neurobehavioral changes of locomotor retardation and cognition and memory disorders in earthworms. Earthworms Cypermethrin Neurobehavioral Neurotoxicity Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Cypermethrin is a widely used pyrethroid insecticide around the world (Tang et al., 2018 ; Xie et al., 2021 ). Due to the fact that cypermethrin is a non-polar insecticide that is easily absorbed and fixed by the soil (Al-Smadi et al., 2019 ; Amin et al., 2021 ), most of the cypermethrin used in agriculture ultimately becomes residues in the soil, which poses a potential risk to soil-dwelling organisms. Earthworm is an important indicator animal of the soil environment. Using earthworm biomarkers to evaluate the harm of pollutants to soil-dwelling organisms, so as to evaluate the risk to the soil ecosystem, has become one of the research hotspots in the field of environmental research (Muangphra et al., 2015 ; Shi et al., 2017 ; Tiwari et al., 2019 ; Guo et al., 2020 ; Mishra et al., 2022 ). Studies have shown that cypermethrin can cause the death of earthworms, and the LC 50 value of cypermethrin to adult earthworms is 61.18–93.81 mg/kg (Zhou et al., 2008 ). When cypermethrin is applied at the manufacturers’ recommended rates, the original deposition of cypermethrin in soil was 1.71 ± 0.31 mg/kg (Zhu et al., 2022 ). Moreover, cypermethrin in the soil can be degraded by microorganisms and light. Therefore, the cypermethrin present as residuals in a field situation is often lower than the lethal dose of cypermethrin to earthworms. Cypermethrin is a neurotoxic pesticide, and the nervous system of organisms is a major target of this pesticide and is very sensitive to cypermethrin (Korytko et al., 1998; Mohammadi et al., 2019 ; Yadav et al., 2021 ). Although the residue of cypermethrin in the field does not easily lead to earthworm death, the residue may cause nerve damage to earthworms. To date, there have been many reports on the chronic toxicity of cypermethrin to earthworms, but these studies mainly focus on the chronic toxicity of cypermethrin to earthworms' growth and reproduction (Zhou et al., 2008 ; Zhou et al., 2011 ; Pelosi et al., 2014 ; Zhu et al., 2022 ; Mishra et al., 2022 ), and rarely involve the study of neurobehavioral disorders in earthworm. Nervous behavior is an integrated neural activity, which can comprehensively reflect the nerve injury of animals, and is an important indicator for evaluating the neurotoxicity of chemicals (Winneke et al., 2007; Gargouri et al., 2018 ; Wang et al., 2019 ; Mastella et al., 2022 ). Studies have shown that cypermethrin has an impact on the normal function of the animal nervous system and causes neurobehavioral disorders in animals (Casco et al., 2006 ; Tian et al., 2009 ; Singh et al., 2012 ; Liu et al., 2019 ; Zhou et al., 2020 ). However, whether the effects on the nervous system of earthworms exposed to low doses of cypermethrin, cause changes in the earthworm's neurobehavior and the resulting ecological impact is a problem that has not been clear in the current research. The studies on these questions would not only help to elucidate the neurotoxic mechanism of cypermethrin to soil animals but also be beneficial to accurately evaluate the risk of neurotoxic pesticide pollutants to soil animals. In this study, we selected Amynthas corticis , a dominant species of earthworm commonly found in China, as the experimental organism and investigated the changes in the nerve behavior of earthworms and the ion channel characteristic enzymes (Ca 2+ -ATP, Mg 2+ -ATP, Ca 2+ -Mg 2+ -ATP, and Na + -K + -ATP ) in the cerebral ganglions (CGs) of earthworms induced by cypermethrin exposure. The subchronic neurotoxicity of cypermethrin in earthworms was investigated by using the integrated biomarker response IBR and molecular docking techniques. This study aimed to test the subchronic neurotoxicity of low-dose cypermethrin on earthworms, to explore the possible mechanisms of the neurological effects of cypermethrin exposure on earthworms, and to provide theoretical basis and technical support for the ecological risk assessment of neurotoxic pesticide contamination. 2. Materials and Methods 2.1. Chemicals and earthworms Cypermethrin (CAS Nos.52315-07-8, purity 98.0%) was provided by Sigma-Aldrich Trading Co., Ltd. (Shanghai, China), and other chemicals used for the toxicity tests were provided by Jinen Chemical Co, Ltd. (Shandong, China). Amynthas corticis were collected from Kunming, Yunnan, China. The identification of Amynthas corticis was based on their morphological characteristics, such as setae and spermathecal pores, etc. According to the Organization for Economic Cooperation and Development (OECD 207, 1984), earthworms were fed with oat flour and cultivated at a room temperature of 20 ± 1°C. 2.2. Subchronic toxicity experiments The subchronic toxicity experiment was adapted from OECD Guidelines 207 (OECD 207,1984), using the natural soil described above instead of artificial soil. The natural soil was collected from the 2–15 cm cultivation layer and used as a medium for sub-chronic toxicity experiments. The soil type was red clay with an organic matter content of 18.06 g/kg, the cation exchange of 9.8 cmol/kg, pH 6.45, and no cypermethrin was detected. In toxicity experiments, the concentration of cypermethrin was respectively set to 2 mg/kg and 6 mg/kg, which is set based on the estimated residue of cypermethrin in the field after application of the manufacturer's recommended application dose. When cypermethrin is applied at the maximum field rate( 90 g/hm 2 ), the original deposition of cypermethrin in soil was 1.71 ± 0.31 mg/kg (Zhu et al., 2022 ). As the frequency of applications of cypermethrin in the field was generally 1–3 times, assuming that cypermethrin in the soil was not degraded, the cumulative deposition of cypermethrin in soil almost equates to 2 mg/kg and 6 mg/kg respectively. The desired amount of cypermethrin was thoroughly mixed into 1000 g of the soil by adding an acetone-cypermethrin solution to achieve the working concentration of cypermethrin. After acetone volatilization, soil moisture was adjusted to 60% water holding capacity using deionized water and contaminated soils were transferred to 2 L incubation bottles and Amynthas corticis (n = 20) added. The incubation bottles were sealed with plastic wrap (with holes reserved for air exchange) to prevent earthworms from escaping. The earthworms were cultured in incubation bottles (20 ℃, 60% humidity) for 56 days. During experiment, 5 g of oat flour was spread on the soil surface to feed earthworms every week. The incubation bottles containing contaminated soil and earthworms were weighed, and the loss of water by evaporation was compensated by the addition of deionized water every 3 d. The control treatment free of cypermethrin was conducted. Each treatment and control had three replicates. 2.3. Determination of biomarkers of exposure 2.3.1. Biomarkers of exposure: neurobehavior On the 28th and 56th of the subchronic toxicity experiment, earthworms were randomly removed from the control group and the treatment group for earthworm cognition, memory, and motor behavior testing. Before the cognition and memory behavior testing for earthworms, earthworms were trained. The training was based on the earthworm's insensitivity to vibration stimuli and aversion to white light stimuli and used white light paired with vibration stimuli to train the earthworm to learn the strategy of stopping the appearance of white light stimuli by accelerating movement. If the time for earthworms to reach the specified threshold after training was less than the time for them to reach the specified threshold before training, it indicated that earthworms had understood the strategy of preventing white light stimulation through accelerated movement. The training and behavior testing of earthworms using established methods (Chen et al., 2021 ). The behavior test device of earthworm is shown in Fig. 1 . To simulate the moving environment of earthworms in the soil, the test bench was covered with a 40×40 cm plastic pad with circular bulges (the height of the circular bulge is 0.5 cm, and the distance between the adjacent bulges is 3 cm), and a vibration motor with a vibration frequency of 180 Hz is set at the bottom. Above the test bench, a spraying device (spraying volume 20 mL/h ) with timer control one red lamp, and one filament lamp of 3 power were installed. The red lamp was used as the lighting source for training and testing so that the behavior of earthworms could be observed, and a bright white lamp served as the aversive light stimulus. The moving displacement threshold of the earthworm was set to 9 cm during training, and each training cycle included 30 s vibration/30 s white light/30 s vibration/30 s white light/30 s vibration/30 s white light. During white light stimulation training, if the earthworm can move to the specified threshold within 30 s, turn off the white light immediately until the end of 30 s and then enter into vibration stimulation. The training of earthworms is 3 cycles. Among them, the earthworm rest recovery time after each training period was 7 min. When the time for earthworms to reach the specified threshold was less than the time for them to reach the specified threshold before training, it indicated that earthworms had grasped the strategy to prevent white light stimulation through accelerated movement, and the training was over. The cognition and memory behavior testing of earthworms was conducted 24 hours after the end of training. The time for the earthworm's head to reach or exceed the movement displacement threshold was detected during the 30 s vibration/30 s white light/30 s vibration/30 s white light/30 s vibration/30 s white light. The earthworm motor response test was performed before the earthworm training, and the conditions were the same as the cognition and memory behavior test conditions except that the training was not required. 2.3.2. Biomarkers of exposure: ion channel characteristic enzymes activities After the earthworm neurobehavioral test was over, earthworms were removed and anesthetized with 0.2% chlorobutanol in water. The cerebral ganglions (CGs) were dissected by using a dissection microscope and stored at -80℃. Ca 2+ -ATP, Mg 2+ -ATP, Ca 2+ -Mg 2+ -ATP, and Na + -K + -ATP activities in the CGs of earthworms were determined with reference to (Zhao et al., 2000 ; Torlinska and Grochowalska, 2004 ). 2.4 Molecular docking studies Ca 2+ -ATPase (5ZTF) of Homo sapiens was downloaded and extracted amino acid sequence from the RCSB PDB database ( https://www.rcsb.org/ ). Downloading the protein sequence of Amynthas corticis from the Genome Warehouse ( https://bigd.big.ac.cn/gwh ) in the National Genomics Data Center (NGDC). Using blastp (E-value 60%) through Ca 2+ -ATPase (5ZTF) as query sequence, the homologous gene of Ca 2+ -ATPase in Amynthas corticis was identified for obtaining the protein sequence of Amynthas corticis . Homology models of Ca 2+ -ATPase of Amynthas corticis were built using the Swiss-Model server, and PDB 5ZTF was used as a template. The model was visualized with PyMOL. Model quality as measured by QMEANDisCo Global score45 was 0.71 ± 0.05 and sequence identity was 71.59%. Molecular docking of Bifenthrin was performed using AutoDock. Show receptor-ligand interaction in a 2D diagram using Discovery Studio Visualize soft. 2.5 Statistical analysis All data were statistically analyzed using SPSS26.0 and Origin Pro8.0 software. One-way analysis of variance (ANOVA; P < 0.05) was performed using SPSS26.0 to determine significant differences between treatments and controls, followed by post hoc tests based on the least significant difference (LSD); one-way ANOVA was used for the analysis of multiple comparisons (LSD) between different concentration groups. Statistical significance was expressed at a probability level of P < 0.05. The integrated biomarker response (IBR) of test organisms exposed to cypermethrin was determined and the corresponding star and bar graphs were generated. Calculations were performed according to the methodology of (Sanchez et al., 2013 ). 3. Results and discussion No mortality was observed during the exposure time. 3.1. Biomarkers of chronic exposure: neural behavior The cognition and memory behavior is a process in which animals encode, store, and extract new information after obtaining new information from the external environment(Shettleworth et al., 2001; Ghafarimoghadam et al., 2022 ; Luis and Ryan., 2022). Since it is impossible to observe the internal cognition and memory process of earthworms directly, the study of the cognition and memory of earthworms can only acquire the coding form, storage capacity, retention time, and dependent conditions of these processes by measuring their operation performance or reaction time after learning and performing a task. The test of cognition and memory behavior of earthworms was designed in this paper based on earthworms' dislike of white light stimulation. Earthworms were trained to understand the strategy of preventing white light stimulation with fast movement, and the information acquisition status of earthworms was evaluated at 24 h after the end of training by measuring the time when earthworms reached the movement displacement threshold during white light stimulus. The effect on the time for earthworms exposed to cypermethrin reaching the movement displacement threshold is shown in Fig. 2 . The time for earthworms reaching the movement displacement threshold in the control groups and all treatment groups after training was significantly lower than that before training during the exposure period ( P < 0.05), it showed that the trained earthworms had understood the strategy of preventing white light stimulation by accelerating their movement. 24 h after the end of training, only in the control group and the pesticide low concentration treatment groups (2 mg/kg cypermethrin ), the time for earthworms to reach the movement displacement threshold was not significantly different from that of the end of training ( P > 0.05), indicating that the earthworms still retained the memory that accelerated movement could prevent the appearance of white light stimulation, and could avoid white light stimulation by accelerating movement during the test. However, the time for earthworms exposed to 6 mg/kg cypermethrin to reach the movement displacement threshold was significantly increased compared with that of the end of training ( P 0.05), it indicated that the earthworms showed memory disabilities, lost the memory that accelerated movement could prevent white light stimulation, and could not avoid white light stimulation by accelerating movement during the test. Although the brain capacity of earthworms is small, it is found that earthworms still have the cognitive behavior, which is consistent with previous studies on earthworms' cognitive behavior. Yerkes first confirmed the learning ability of earthworm ( Allolobophora foetida ) through experiments (Yerkes, 1912 ). Subsequent studies also showed that earthworms of different species such as Lumbricus terrestris and Eisenia foetida have certain cognition and memory behavior (Abramson and Buckbee., 1995; Li and Pan., 2008). In the present study, 24 h after the end of training, the earthworms in the control groups and 2 mg/kg cypermethrin groups retained the memory that accelerated movement could prevent the appearance of white light stimulation, and could avoid white light stimulation by accelerating movement during the test, but the earthworms exposed to 6 mg/kg cypermethrin could not avoid white light stimulation by accelerating movement due to memory disabilities, which led to a significant increase in the time for earthworms reaching the movement displacement threshold compared with that of the end of training ( P < 0.05 ). Cypermethrin not only affected cognition and memory behavior in earthworms but also adversely affected earthworm movement. As shown in Fig. 2 ., in all treatment groups during the exposure period, the time to reach the moving displacement threshold for earthworms was significantly higher than that of the control group, either before or after training ( P < 0.05 ). It showed that the rate of movement for earthworms in all treatments was slower compared with the control group, and cypermethrin caused abnormal changes in motor behavior for earthworms such as slowness of movement. Cognition, memory, and motor behavior are dominated by the nervous system, and the behavior changes can comprehensively reflect the nerve injury of animals(Pessoa et al., 2019 ; Takagi and Benton., 2020; Giordano et al., 2021 ; Coria-Avila et al., 2022 ). In this study, the abnormal changes in motor, cognition, and memory behavior in earthworms exposed to cypermethrin showed that the nerves of earthworms were damaged. The cognition and memory disorder of earthworms will make it impossible for earthworms to store or extract the information obtained, and it is difficult for earthworms to make adaptive changes with the help of experiences, which is not conducive to the survival and development of earthworms. Further, the motor is a behavior controlled by the nervous system, which is the basis of earthworm survival. Damage to the nerve of earthworms can lead to sluggish movement of earthworms, making it difficult for earthworms to find food quickly, avoid natural enemies, and reduce their range of activity, which is not conducive to earthworms occupying richer resources and more suitable living space. 3.2. Biomarkers of exposure: ion channel characteristic enzyme activities in the CGs of earthworms Cerebral ganglions (CGs) are the structures linked to the nerve cord of the supraphrayngeal ganglions and involve many functions like motor control, emotion, and cognition(Subaraja and Vanisree, 2019 ). CGs in earthworm are a set of nerve rings and it is located over the pharynx in the body of 3rd segments and connects with the ventrally located subpharyngeal one (Carew and Sahley, 1986 ; Subaraja and Vanisree, 2019 ). The effect on Na + -K + -ATPase activity in the CGs of earthworms exposed to cypermethrin is shown in Fig. 3 A. During the exposure period, Na + -K + -ATPase activity in the CGs of earthworms was inhibited by exposure to 2 and 6 mg/kg cypermethrin compared with controls ( P < 0.05), and there was difference between activity at the two concentrations ( P < 0.05). At 56 d of exposure, Na + -K + -ATPase activity in earthworms exposed to cypermethrin was significantly lower than those in the CGs of earthworms at 28 d of exposure( P < 0.05). It showed that cypermethrin exposure significantly inhibited the Na + -K + -ATPase activity in the CGs of earthworms, the inhibition effect increased with the increase of exposure time, the greater the concentration of cypermethrin exposure, and the greater the inhibition of Na + -K + -ATPase activity in the CGs of earthworms. Na + -K + -ATPase catalyzes the ATP hydrolysis reaction, releasing the free energy stored in ATP for use by the organism, especially driving the active transport of Na + and K + (Villar, 2019 ). Due to the high energy required for motion, Na + - K + - ATPase activity is crucial for maintaining normal motor function in animals (Yorek et al., 1993 ; Haddar et al., 2018 ). In the present study, although no earthworm poisoning death occurred in all cypermethrin-treated groups, earthworms showed a decline in the rate of movement compared with the control group, which was consistent with the change of Na + - K + - ATPase activity in the CGs of earthworms. During the exposure period, Ca 2+ -ATPase, Ca 2+ -Mg 2+ -ATPase and Mg 2+ -ATPase activities in the CGs of earthworms for 6 mg/kg cypermethrin were significantly lower than that of the control, but there were no difference in Ca 2+ -ATPase (Fig. 3 B), Ca 2+ -Mg 2+ -ATPase (Fig. 3 C) and Mg 2+ -ATPase (Fig. 3 D) activities between the control and 2 mg/kg cypermethrin ( P > 0.05). It showed that the effect of cypermethrin on Ca 2+ -ATPase, Ca 2+ -Mg 2+ -ATPase, and Mg 2+ -ATPase activities in the CGs of earthworms depended on the exposure concentration, and Ca 2+ -ATPase, Ca 2+ -Mg 2+ -ATPase, and Mg 2+ -ATPase activities were significantly inhibited by cypermethrin only at the higher concentration (6 mg/kg). The inhibition of Ca 2+ -ATPase, Ca 2+ -Mg 2+ -ATPase, and Mg 2+ -ATPase activities in the CGs of earthworms by cypermethrin will lead to abnormal Ca 2+ distribution inside and outside the neural membrane. Under normal conditions, the distribution of Ca 2+ inside and outside the neural membrane is different, with the extra-membrane being higher than the intra-membrane, and the regulation of Ca 2+ inside and outside the membrane is mainly done by Ca 2+ -ATPase, Ca 2+ -Mg 2+ -ATPase, and Mg 2+ -ATPase (Jilka et al., 1975 ; Hogaboom and Fedan., 1980; Weber et al., 2021 ). Ca 2+ -ATPase can catalyze ATP hydrolysis on the inner side of the plasma membrane, release energy, and drive intracellular calcium ions to pump out of cells or pump them into the lumen of the endoplasmic reticulum for storage, so as to maintain a low concentration of free Ca 2+ in cells (Choi, 1995 ). Because its activity depends on the combination of ATP and Mg 2+ , it forms a calcium pump together with Ca 2+ -Mg 2+ -ATPase, which is an important basis for maintaining low Ca 2+ homeostasis in nerve cells (Weber et al., 2021 ). Mg 2+ -ATPase is also an ion pump, providing energy for the active transport of Mg 2+ against the concentration gradient, thereby maintaining a relatively constant Mg 2+ concentration and osmotic pressure inside and outside the cell membrane(Tomita et al., 2017 ; Tashiro et al., 2019 ). The inhibition of Ca 2+ -ATPase and Ca 2+ -Mg 2+ -ATPase activities in the CGs of earthworms by cypermethrin will lead to abnormal distribution of Ca 2+ inside and outside the nerve membrane, causing intracellular calcium overload. At the same time, the decrease of Mg 2+ -ATPase activity may cause the decline of intracellular Mg 2+ , weaken the physiological antagonism to the increase of Ca 2+ , and aggravate calcium overload. Since Ca 2+ plays an important role in the induction and maintenance of hippocampal long-term potentiation (LTP), a synaptic model of cognition and memory function (Smith, 1987 ; Miyamoto and Fukunaga., 1996; Devine et al., 2022 ), the abnormal Ca 2+ distribution inside and outside the neural membrane and intracellular calcium overload will affect the cognition and memory behavior of earthworms. In this study, the cognition and memory disabilities in earthworms were observed only at 6 mg/kg cypermethrin-treated groups, Ca 2+ -ATPase, Ca 2+ -Mg 2+ -ATPase, and Mg 2+ -ATPase activities in earthworms exposure to 6 mg/kg cypermethrin were inhibited compared with controls ( P 0.05), indicating that the intracellular calcium overload caused by inhibition of Ca 2+ -ATPase, Ca 2+ -Mg 2+ -ATPase and Mg 2+ -ATPase activities may be one of the mechanisms of cognition and memory impairment in earthworms induced by cypermethrin exposure. 3.3. Binding model of cypermethrin and Ca 2+ -ATPase Given that the abnormal Ca 2+ distribution inside and outside the nerve membrane of earthworms triggered by cypermethrin exposure may be a possible mechanism leading to neurobehavioral changes such as cognitive deficits in earthworms, and Ca 2+ -ATP is one of the main enzymes determining the Ca 2+ distribution inside and outside the nerve membrane of earthworms, the Ca 2+ -ATPase was chosen as the receptor for molecular docking to explore the molecular mechanism of the neurotoxicity of cypermethrin in earthworms (Colina et al., 2002 ; Qi et al., 2021 ; Zhang et al., 2022 ). The three-dimensional structure of the Ca 2+ -ATPase receptor of the earthworm needs to be obtained using homology modeling. A PDB sequence number 5ZTF (Fig. 4 .), which is known and has the highest homology to the Ca 2+ -ATPase primary sequence, was selected as a template for homology modeling. The homology similarity between the study subject Amynthas corticis Ca 2+ -ATPase (GWHGAOSM004429.1) and 5ZTF was 71.59%. The Ramachandran plot (Fig. 5 .) shows that the percentage of GWHGAOSM004429.1 protease residues falling into the most favorable region is 95.49%, while the percentage of residues located in the region not in the active site are all less than 4.51%, indicating that the structure of the Ca 2+ -ATP protein model modeled by using the 5ZTF as a template homology is in general reasonable and can be used for molecular docking. The results of molecular docking of GWHGAOSM004429.1 with cypermethrin are shown in Fig. 6. Residues LYS-728 and ALA-725 in GWHGAOSM004429.1 interacted with cypermethrin to form two hydrogen bonds with lengths of 2.0 and 2.7A, respectively. The binding energy of GWHGAOSM004429.1 with cypermethrin was − 8.17 kcal/mol, suggesting that cypermethrin interacts with Ca 2+ -ATP receptor proteins and that this interaction occurs spontaneously and a lower energy and more stable complex is obtained, the formation of which consumes Ca 2+ -ATPase, resulting in a decrease in Ca 2+ -ATPase content and a decrease in enzyme activity. In this study, Fig. 3 B showed that cypermethrin exposure inhibited Ca 2+ -ATPase activity within earthworm CGs, which is consistent with the results of the molecular docking of cypermethrin and Ca 2+ -ATP receptor proteins. The results of molecular docking of cypermethrin with Ca 2+ -ATPase showed that cypermethrin inhibited earthworm Ca 2+ -ATPase activity by interacting with Ca 2+ -ATPase to form a stable complex. With the formation of this complex, the Ca 2+ -ATPase content in the CGs of earthworms continued to decrease, which triggered the abnormal distribution of Ca 2+ inside and outside the nerve membranes of earthworms, and the intracellular calcium overload, which led to neurobehavioral changes, such as cognition and memory deficits in earthworms. 3.4. Comparison of subchronic neurotoxicity using the integrated biomarker response index The IBR method is a comprehensive evaluation tool to assess integrated toxicity effects in an organism(Hou et al., 2016 ; Xu et al., 2020 ; Wang et al., 2022 ). In the present study, responses from six biomarkers were normalized and expressed as star plots using exposure values for the 2, 6 mg/kg cypermethrin amendments on 28 d and 56 d (Fig. 7 .). Since the IBR was the sum of normalized values for all indicators, the larger the normalized value, the bigger the influence of pesticides on earthworm (Normalized value greater than 0 means activated, less than 0 means suppressed). In addition, the IBR values for cypermethrin are also presented in bar chart format (Fig. 8 .) with larger values indicating greater neurotoxicity. As shown in Fig. 8 ., the IBR values for 2, 6 mg/kg cypermethrin were 1.46, and 4.78 at 28 d, respectively, indicating that the neurotoxicity of cypermethrin to earthworms depended on the exposure concentration, the more exposure concentration, the greater of neurotoxicity of cypermethrin to earthworms at 28 d. With increased exposure time, the IBR values for 2, 6 mg/kg cypermethrin were 2.61, and 7.04 at 56 d, respectively, the IBR value for cypermethrin at 56 d was greater than that of the same concentration at 28 d, indicating that the subchronic neurotoxicity of cypermethrin to earthworms increased with increasing exposure time. 4. Conclusion The results of the subchronic neurotoxicity study of earthworms exposed to cypermethrin showed that although the chronic neurotoxicity of cypermethrin did not lead to the death of earthworms, it induced neurobehavioral changes, such as locomotor retardation and cognitive deficits in earthworms. The inhibition of Ca 2+ -ATPase activity in earthworms triggered by the interaction of cypermethrin with Ca 2+ -ATPase to form a stable complex is one of the possible molecular mechanisms of cognitive impairment in earthworms. The subchronic neurotoxicity of cypermethrin for earthworms increased with the increase of the exposure concentration and the duration of cypermethrin exposure, and the subchronic neurotoxicity of cypermethrin to earthworms leads to neurological damage in earthworms, but whether this neurological damage recovers with the elimination of the exposure remains to be further investigated. Declarations Acknowledgments This work was supported by the National Science Foundations of China (No. 31860155), the Yunnan Natural Sciences Foundations (No.2017FG001(-040)), and the Natural Sciences Foundations of Key Laboratory of State Forestry Administration on Highly-Efficient Utilization of Forestry Biomass Resources in Southwest China (No. 2022-KF09). Ethical approval and consent to participate Not applicable (this article does not contain studies involving human participants or their tissues) Consent to publish Not applicable Authors contributions FuHui Zhao : Conceptualization, Formal analysis, Investigation, Methodology, Data curation, Writing-original draft. Sijia Wu : Conceptualization, Visualization, Resources, Formal analysis, Investigation, Writing-original draft. ShiPing Zhou : Conceptualization, Validation, Writing-review & editing, Project administration, Funding acquisition. HuiJuan Li : Investigation, Formal analysis, Resources. QiSheng Li : Investigation, Formal analysis, Resources. ShouQing Liu : Investigation, Methodology, Writing-review & editing. HuaYin Liu: Software, Formal analysis, Resources. Mei Qin : Formal analysis, Data Curation, Supervision. Competing interests The authors declare no competing interests. References Abramson, C.I., Buckbee, D.A., 1995. 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Yorek, M.A., Wiese, T.J., Davidson, E.P., Dunlap, J.A., Stefani, M.R., Conner, C.E., Lattimer, S.A., Kamijo, M., Greene, D.A., Sima, A.A.F., 1993. Reduced motor nerve conduction velocity and Na + -K + -ATPase activity in rats maintained on L-Fucose diet: Reversal by myo-Inositol supplementation . Diabetes. 42, 1401–1406. Tian, Y.T., Liu, Z.W., Yao, Y., Yang, Z., Zhang, T., 2009. Effect of alpha-cypermethrin and theta-cypermethrin on delayed rectifier potassium currents in rat hippocampal neurons. Neurotoxicology. 30, 269-273. Zhang, W., Ye, F.H., Pang, N., Kessi, M., Xiong, J., Chen, S.M., Peng, J., Yang, L., Yin, F., 2022. Restoration of sarco/endoplasmic reticulum Ca 2+ -ATPase activity functions as a pivotal therapeutic target of anti-glutamate-induced excitotoxicity to attenuate endoplasmic reticulum Ca 2+ depletion. Front. Pharmacol. 13, 877175. Zhao, Y.L., Song, J.P.,Yang, Y.H., Ma, H.B., Pu, J.S., 2000. Effects of microwave radiation of different intensities on the activity of Ca 2+ , Mg 2+ -ATPase in mouse brain tissues. Chinese J. Aerosp. Med. 11, 101–104. Zhou, L.H., Zhou, M.Q., Tao, H.D., Xiao, M.X., 2020. Cypermethrin-induced cortical neurons apoptosis via the Nrf2/ARE signaling pathway. Pestic. Biochem. Physiol. 165, 02.013. Zhou, S.P., Duan, C.Q., Michelle, W.H.G., Yang, F.Z., Wang, X.H., 2011. Individual and combined toxic effects of cypermethrin and chlorpyrifos on earthworm. J. Environ. Sci. 23, 676-680. Zhou, S.P., Duan, C.Q., Wang, X.H., Michelle, W.H.G., Yu, F.Z., Fu, H., 2008. Assessing cypermethrin-contaminated soil with three different earthworm test methods. J. Environ. Sci. 20, 1381-1385. Zhu, X.Z., Xiong, Z.P., Zhou, S.P., Xie, S.D., Li,H. J., Li, Q.S., Yang, G.B., 2022. Analysis of reproductive damage in earthworms ( Amynthas corticis ) exposed to cypermethrin. Ecotoxicol. Environ. Saf. 244, 114038. 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Values are presented as the mean±SD (n = 3). Different letters above columns indicate significant differences at \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 level between treatments.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4097539/v1/ba038c6c76de4527305475bc.png"},{"id":54847857,"identity":"d2dd5600-5cd2-4c2d-881c-c46989ae3a99","added_by":"auto","created_at":"2024-04-17 15:28:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51697,"visible":true,"origin":"","legend":"\u003cp\u003eIon channel characteristic enzymes activities in the CGs of earthworms after exposure to cypermethrin for 28 and 56 d. Different small letters above the column indicate that there was a significant difference in the enzymes activities in earthworms in the same concentration treatment groups between different exposure times \u003cem\u003e(P\u0026lt; 0.05)\u003c/em\u003e, and different capital letters indicate that there was a significant difference in the enzymes activities in different concentration treatment groups under the same exposure time\u003cem\u003e(P\u0026lt; 0.05).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4097539/v1/dc73c6dd8e6c400f23f34e35.png"},{"id":54848389,"identity":"fb52a88c-5209-48ee-a9a7-62fc9e728e63","added_by":"auto","created_at":"2024-04-17 15:36:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":322214,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of the template 5ZTF\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4097539/v1/146cefece7255187fccbefa0.png"},{"id":54847856,"identity":"498aec9d-fea3-4749-b8d1-4a1593e68b80","added_by":"auto","created_at":"2024-04-17 15:28:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":349942,"visible":true,"origin":"","legend":"\u003cp\u003eRamachandran plot of GWHGAOSM004429.1 protease with 5ZTF homology modeling\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4097539/v1/60dbe168b523d2bfd08199e8.png"},{"id":54847861,"identity":"99bd58d3-aef2-46ce-a2bb-f6e937ba7dbf","added_by":"auto","created_at":"2024-04-17 15:28:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":425012,"visible":true,"origin":"","legend":"\u003cp\u003eA. Molecular docking simulation of cypermethrin small molecules with Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase; B. 3D plot of molecular docking of cypermethrin with Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase; C. 2D plot of molecular docking of cypermethrin with Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4097539/v1/af4875beac71f19f205e758b.png"},{"id":54847859,"identity":"3c8e5f8c-8e83-478b-bdc1-17703f7c6265","added_by":"auto","created_at":"2024-04-17 15:28:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":223941,"visible":true,"origin":"","legend":"\u003cp\u003eBiomarker response analysis of earthworm neurotoxicity.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4097539/v1/8a5e6c30c85443abb4e286b9.png"},{"id":54848390,"identity":"0cfddb0a-616d-4d58-93d8-caacd6c3be1a","added_by":"auto","created_at":"2024-04-17 15:36:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":23457,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in IBR index of neurotoxicity of cypermethrin to earthworms\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4097539/v1/c159374593ae4d76930297b8.png"},{"id":67986223,"identity":"2683278b-9aa2-485d-969a-00d691bbf4a2","added_by":"auto","created_at":"2024-11-01 04:05:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1990380,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4097539/v1/6e998631-c53b-4566-abc4-33722c02181b.pdf"}],"financialInterests":"","formattedTitle":"Subchronic Neurotoxic Effects of Cypermethrin on Earthworms","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCypermethrin is a widely used pyrethroid insecticide around the world (Tang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Due to the fact that cypermethrin is a non-polar insecticide that is easily absorbed and fixed by the soil (Al-Smadi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Amin et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), most of the cypermethrin used in agriculture ultimately becomes residues in the soil, which poses a potential risk to soil-dwelling organisms.\u003c/p\u003e \u003cp\u003eEarthworm is an important indicator animal of the soil environment. Using earthworm biomarkers to evaluate the harm of pollutants to soil-dwelling organisms, so as to evaluate the risk to the soil ecosystem, has become one of the research hotspots in the field of environmental research (Muangphra et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shi et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tiwari et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mishra et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies have shown that cypermethrin can cause the death of earthworms, and the LC\u003csub\u003e50\u003c/sub\u003e value of cypermethrin to adult earthworms is 61.18\u0026ndash;93.81 mg/kg (Zhou et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). When cypermethrin is applied at the manufacturers\u0026rsquo; recommended rates, the original deposition of cypermethrin in soil was 1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 mg/kg (Zhu et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, cypermethrin in the soil can be degraded by microorganisms and light. Therefore, the cypermethrin present as residuals in a field situation is often lower than the lethal dose of cypermethrin to earthworms.\u003c/p\u003e \u003cp\u003eCypermethrin is a neurotoxic pesticide, and the nervous system of organisms is a major target of this pesticide and is very sensitive to cypermethrin (Korytko et al., 1998; Mohammadi et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yadav et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although the residue of cypermethrin in the field does not easily lead to earthworm death, the residue may cause nerve damage to earthworms. To date, there have been many reports on the chronic toxicity of cypermethrin to earthworms, but these studies mainly focus on the chronic toxicity of cypermethrin to earthworms' growth and reproduction (Zhou et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pelosi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mishra et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and rarely involve the study of neurobehavioral disorders in earthworm.\u003c/p\u003e \u003cp\u003eNervous behavior is an integrated neural activity, which can comprehensively reflect the nerve injury of animals, and is an important indicator for evaluating the neurotoxicity of chemicals (Winneke et al., 2007; Gargouri et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mastella et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Studies have shown that cypermethrin has an impact on the normal function of the animal nervous system and causes neurobehavioral disorders in animals (Casco et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, whether the effects on the nervous system of earthworms exposed to low doses of cypermethrin, cause changes in the earthworm's neurobehavior and the resulting ecological impact is a problem that has not been clear in the current research. The studies on these questions would not only help to elucidate the neurotoxic mechanism of cypermethrin to soil animals but also be beneficial to accurately evaluate the risk of neurotoxic pesticide pollutants to soil animals.\u003c/p\u003e \u003cp\u003eIn this study, we selected \u003cem\u003eAmynthas corticis\u003c/em\u003e, a dominant species of earthworm commonly found in China, as the experimental organism and investigated the changes in the nerve behavior of earthworms and the ion channel characteristic enzymes (Ca\u003csup\u003e2+\u003c/sup\u003e-ATP, Mg\u003csup\u003e2+\u003c/sup\u003e-ATP, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATP, and Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATP ) in the cerebral ganglions (CGs) of earthworms induced by cypermethrin exposure. The subchronic neurotoxicity of cypermethrin in earthworms was investigated by using the integrated biomarker response IBR and molecular docking techniques. This study aimed to test the subchronic neurotoxicity of low-dose cypermethrin on earthworms, to explore the possible mechanisms of the neurological effects of cypermethrin exposure on earthworms, and to provide theoretical basis and technical support for the ecological risk assessment of neurotoxic pesticide contamination.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and earthworms\u003c/h2\u003e \u003cp\u003eCypermethrin (CAS Nos.52315-07-8, purity 98.0%) was provided by Sigma-Aldrich Trading Co., Ltd. (Shanghai, China), and other chemicals used for the toxicity tests were provided by Jinen Chemical Co, Ltd. (Shandong, China).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmynthas corticis\u003c/em\u003e were collected from Kunming, Yunnan, China. The identification of \u003cem\u003eAmynthas corticis\u003c/em\u003e was based on their morphological characteristics, such as setae and spermathecal pores, etc. According to the Organization for Economic Cooperation and Development (OECD 207, 1984), earthworms were fed with oat flour and cultivated at a room temperature of 20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Subchronic toxicity experiments\u003c/h2\u003e \u003cp\u003eThe subchronic toxicity experiment was adapted from OECD Guidelines 207 (OECD 207,1984), using the natural soil described above instead of artificial soil. The natural soil was collected from the 2\u0026ndash;15 cm cultivation layer and used as a medium for sub-chronic toxicity experiments. The soil type was red clay with an organic matter content of 18.06 g/kg, the cation exchange of 9.8 cmol/kg, pH 6.45, and no cypermethrin was detected. In toxicity experiments, the concentration of cypermethrin was respectively set to 2 mg/kg and 6 mg/kg, which is set based on the estimated residue of cypermethrin in the field after application of the manufacturer's recommended application dose. When cypermethrin is applied at the maximum field rate( 90 g/hm\u003csup\u003e2\u003c/sup\u003e), the original deposition of cypermethrin in soil was 1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 mg/kg (Zhu et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As the frequency of applications of cypermethrin in the field was generally 1\u0026ndash;3 times, assuming that cypermethrin in the soil was not degraded, the cumulative deposition of cypermethrin in soil almost equates to 2 mg/kg and 6 mg/kg respectively.\u003c/p\u003e \u003cp\u003eThe desired amount of cypermethrin was thoroughly mixed into 1000 g of the soil by adding an acetone-cypermethrin solution to achieve the working concentration of cypermethrin. After acetone volatilization, soil moisture was adjusted to 60% water holding capacity using deionized water and contaminated soils were transferred to 2 L incubation bottles and \u003cem\u003eAmynthas corticis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;20) added. The incubation bottles were sealed with plastic wrap (with holes reserved for air exchange) to prevent earthworms from escaping. The earthworms were cultured in incubation bottles (20 ℃, 60% humidity) for 56 days. During experiment, 5 g of oat flour was spread on the soil surface to feed earthworms every week. The incubation bottles containing contaminated soil and earthworms were weighed, and the loss of water by evaporation was compensated by the addition of deionized water every 3 d. The control treatment free of cypermethrin was conducted. Each treatment and control had three replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Determination of biomarkers of exposure\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Biomarkers of exposure: neurobehavior\u003c/h2\u003e \u003cp\u003eOn the 28th and 56th of the subchronic toxicity experiment, earthworms were randomly removed from the control group and the treatment group for earthworm cognition, memory, and motor behavior testing. Before the cognition and memory behavior testing for earthworms, earthworms were trained. The training was based on the earthworm's insensitivity to vibration stimuli and aversion to white light stimuli and used white light paired with vibration stimuli to train the earthworm to learn the strategy of stopping the appearance of white light stimuli by accelerating movement. If the time for earthworms to reach the specified threshold after training was less than the time for them to reach the specified threshold before training, it indicated that earthworms had understood the strategy of preventing white light stimulation through accelerated movement.\u003c/p\u003e \u003cp\u003eThe training and behavior testing of earthworms using established methods (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The behavior test device of earthworm is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. To simulate the moving environment of earthworms in the soil, the test bench was covered with a 40\u0026times;40 cm plastic pad with circular bulges (the height of the circular bulge is 0.5 cm, and the distance between the adjacent bulges is 3 cm), and a vibration motor with a vibration frequency of 180 Hz is set at the bottom. Above the test bench, a spraying device (spraying volume 20 mL/h ) with timer control one red lamp, and one filament lamp of 3 power were installed. The red lamp was used as the lighting source for training and testing so that the behavior of earthworms could be observed, and a bright white lamp served as the aversive light stimulus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe moving displacement threshold of the earthworm was set to 9 cm during training, and each training cycle included 30 s vibration/30 s white light/30 s vibration/30 s white light/30 s vibration/30 s white light. During white light stimulation training, if the earthworm can move to the specified threshold within 30 s, turn off the white light immediately until the end of 30 s and then enter into vibration stimulation. The training of earthworms is 3 cycles. Among them, the earthworm rest recovery time after each training period was 7 min.\u003c/p\u003e \u003cp\u003eWhen the time for earthworms to reach the specified threshold was less than the time for them to reach the specified threshold before training, it indicated that earthworms had grasped the strategy to prevent white light stimulation through accelerated movement, and the training was over. The cognition and memory behavior testing of earthworms was conducted 24 hours after the end of training. The time for the earthworm's head to reach or exceed the movement displacement threshold was detected during the 30 s vibration/30 s white light/30 s vibration/30 s white light/30 s vibration/30 s white light. The earthworm motor response test was performed before the earthworm training, and the conditions were the same as the cognition and memory behavior test conditions except that the training was not required.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Biomarkers of exposure: ion channel characteristic enzymes activities\u003c/h2\u003e \u003cp\u003eAfter the earthworm neurobehavioral test was over, earthworms were removed and anesthetized with 0.2% chlorobutanol in water. The cerebral ganglions (CGs) were dissected by using a dissection microscope and stored at -80℃. Ca\u003csup\u003e2+\u003c/sup\u003e-ATP, Mg\u003csup\u003e2+\u003c/sup\u003e-ATP, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATP, and Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATP activities in the CGs of earthworms were determined with reference to (Zhao et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Torlinska and Grochowalska, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Molecular docking studies\u003c/h2\u003e \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e-ATPase (5ZTF) of Homo sapiens was downloaded and extracted amino acid sequence from the RCSB PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Downloading the protein sequence of \u003cem\u003eAmynthas corticis\u003c/em\u003e from the Genome Warehouse (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bigd.big.ac.cn/gwh\u003c/span\u003e\u003cspan address=\"https://bigd.big.ac.cn/gwh\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in the National Genomics Data Center (NGDC). Using blastp (E-value\u0026thinsp;\u0026lt;\u0026thinsp;1e-30, identity\u0026thinsp;\u0026gt;\u0026thinsp;60%) through Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase (5ZTF) as query sequence, the homologous gene of Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase in \u003cem\u003eAmynthas corticis\u003c/em\u003e was identified for obtaining the protein sequence of \u003cem\u003eAmynthas corticis\u003c/em\u003e. Homology models of Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase of \u003cem\u003eAmynthas corticis\u003c/em\u003e were built using the Swiss-Model server, and PDB 5ZTF was used as a template. The model was visualized with PyMOL. Model quality as measured by QMEANDisCo Global score45 was 0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 and sequence identity was 71.59%. Molecular docking of Bifenthrin was performed using AutoDock. Show receptor-ligand interaction in a 2D diagram using Discovery Studio Visualize soft.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were statistically analyzed using SPSS26.0 and Origin Pro8.0 software. One-way analysis of variance (ANOVA; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was performed using SPSS26.0 to determine significant differences between treatments and controls, followed by post hoc tests based on the least significant difference (LSD); one-way ANOVA was used for the analysis of multiple comparisons (LSD) between different concentration groups. Statistical significance was expressed at a probability level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eThe integrated biomarker response (IBR) of test organisms exposed to cypermethrin was determined and the corresponding star and bar graphs were generated. Calculations were performed according to the methodology of (Sanchez et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eNo mortality was observed during the exposure time.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Biomarkers of chronic exposure: neural behavior\u003c/h2\u003e \u003cp\u003eThe cognition and memory behavior is a process in which animals encode, store, and extract new information after obtaining new information from the external environment(Shettleworth et al., 2001; Ghafarimoghadam et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Luis and Ryan., 2022). Since it is impossible to observe the internal cognition and memory process of earthworms directly, the study of the cognition and memory of earthworms can only acquire the coding form, storage capacity, retention time, and dependent conditions of these processes by measuring their operation performance or reaction time after learning and performing a task.\u003c/p\u003e \u003cp\u003eThe test of cognition and memory behavior of earthworms was designed in this paper based on earthworms' dislike of white light stimulation. Earthworms were trained to understand the strategy of preventing white light stimulation with fast movement, and the information acquisition status of earthworms was evaluated at 24 h after the end of training by measuring the time when earthworms reached the movement displacement threshold during white light stimulus.\u003c/p\u003e \u003cp\u003eThe effect on the time for earthworms exposed to cypermethrin reaching the movement displacement threshold is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The time for earthworms reaching the movement displacement threshold in the control groups and all treatment groups after training was significantly lower than that before training during the exposure period (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), it showed that the trained earthworms had understood the strategy of preventing white light stimulation by accelerating their movement. 24 h after the end of training, only in the control group and the pesticide low concentration treatment groups (2 mg/kg cypermethrin ), the time for earthworms to reach the movement displacement threshold was not significantly different from that of the end of training (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that the earthworms still retained the memory that accelerated movement could prevent the appearance of white light stimulation, and could avoid white light stimulation by accelerating movement during the test. However, the time for earthworms exposed to 6 mg/kg cypermethrin to reach the movement displacement threshold was significantly increased compared with that of the end of training (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), there was no significant difference from the time for earthworms to reach the movement displacement threshold before training (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), it indicated that the earthworms showed memory disabilities, lost the memory that accelerated movement could prevent white light stimulation, and could not avoid white light stimulation by accelerating movement during the test.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough the brain capacity of earthworms is small, it is found that earthworms still have the cognitive behavior, which is consistent with previous studies on earthworms' cognitive behavior. Yerkes first confirmed the learning ability of earthworm (\u003cem\u003eAllolobophora foetida\u003c/em\u003e) through experiments (Yerkes, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1912\u003c/span\u003e). Subsequent studies also showed that earthworms of different species such as \u003cem\u003eLumbricus terrestris\u003c/em\u003e and \u003cem\u003eEisenia foetida\u003c/em\u003e have certain cognition and memory behavior (Abramson and Buckbee., 1995; Li and Pan., 2008). In the present study, 24 h after the end of training, the earthworms in the control groups and 2 mg/kg cypermethrin groups retained the memory that accelerated movement could prevent the appearance of white light stimulation, and could avoid white light stimulation by accelerating movement during the test, but the earthworms exposed to 6 mg/kg cypermethrin could not avoid white light stimulation by accelerating movement due to memory disabilities, which led to a significant increase in the time for earthworms reaching the movement displacement threshold compared with that of the end of training (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Cypermethrin not only affected cognition and memory behavior in earthworms but also adversely affected earthworm movement. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e., in all treatment groups during the exposure period, the time to reach the moving displacement threshold for earthworms was significantly higher than that of the control group, either before or after training (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). It showed that the rate of movement for earthworms in all treatments was slower compared with the control group, and cypermethrin caused abnormal changes in motor behavior for earthworms such as slowness of movement.\u003c/p\u003e \u003cp\u003eCognition, memory, and motor behavior are dominated by the nervous system, and the behavior changes can comprehensively reflect the nerve injury of animals(Pessoa et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Takagi and Benton., 2020; Giordano et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Coria-Avila et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, the abnormal changes in motor, cognition, and memory behavior in earthworms exposed to cypermethrin showed that the nerves of earthworms were damaged. The cognition and memory disorder of earthworms will make it impossible for earthworms to store or extract the information obtained, and it is difficult for earthworms to make adaptive changes with the help of experiences, which is not conducive to the survival and development of earthworms. Further, the motor is a behavior controlled by the nervous system, which is the basis of earthworm survival. Damage to the nerve of earthworms can lead to sluggish movement of earthworms, making it difficult for earthworms to find food quickly, avoid natural enemies, and reduce their range of activity, which is not conducive to earthworms occupying richer resources and more suitable living space.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Biomarkers of exposure: ion channel characteristic enzyme activities in the CGs of earthworms\u003c/h2\u003e \u003cp\u003eCerebral ganglions (CGs) are the structures linked to the nerve cord of the supraphrayngeal ganglions and involve many functions like motor control, emotion, and cognition(Subaraja and Vanisree, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). CGs in earthworm are a set of nerve rings and it is located over the pharynx in the body of 3rd segments and connects with the ventrally located subpharyngeal one (Carew and Sahley, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Subaraja and Vanisree, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The effect on Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATPase activity in the CGs of earthworms exposed to cypermethrin is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. During the exposure period, Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATPase activity in the CGs of earthworms was inhibited by exposure to 2 and 6 mg/kg cypermethrin compared with controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and there was difference between activity at the two concentrations (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 56 d of exposure, Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATPase activity in earthworms exposed to cypermethrin was significantly lower than those in the CGs of earthworms at 28 d of exposure(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It showed that cypermethrin exposure significantly inhibited the Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATPase activity in the CGs of earthworms, the inhibition effect increased with the increase of exposure time, the greater the concentration of cypermethrin exposure, and the greater the inhibition of Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATPase activity in the CGs of earthworms.\u003c/p\u003e \u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e-ATPase catalyzes the ATP hydrolysis reaction, releasing the free energy stored in ATP for use by the organism, especially driving the active transport of Na\u003csup\u003e+\u003c/sup\u003e and K\u003csup\u003e+\u003c/sup\u003e (Villar, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Due to the high energy required for motion, Na\u003csup\u003e+\u003c/sup\u003e- K\u003csup\u003e+\u003c/sup\u003e- ATPase activity is crucial for maintaining normal motor function in animals (Yorek et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Haddar et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, although no earthworm poisoning death occurred in all cypermethrin-treated groups, earthworms showed a decline in the rate of movement compared with the control group, which was consistent with the change of Na\u003csup\u003e+\u003c/sup\u003e- K\u003csup\u003e+\u003c/sup\u003e- ATPase activity in the CGs of earthworms.\u003c/p\u003e \u003cp\u003eDuring the exposure period, Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase and Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase activities in the CGs of earthworms for 6 mg/kg cypermethrin were significantly lower than that of the control, but there were no difference in Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) activities between the control and 2 mg/kg cypermethrin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). It showed that the effect of cypermethrin on Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase, and Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase activities in the CGs of earthworms depended on the exposure concentration, and Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase, and Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase activities were significantly inhibited by cypermethrin only at the higher concentration (6 mg/kg).\u003c/p\u003e \u003cp\u003eThe inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase, and Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase activities in the CGs of earthworms by cypermethrin will lead to abnormal Ca\u003csup\u003e2+\u003c/sup\u003e distribution inside and outside the neural membrane. Under normal conditions, the distribution of Ca\u003csup\u003e2+\u003c/sup\u003e inside and outside the neural membrane is different, with the extra-membrane being higher than the intra-membrane, and the regulation of Ca\u003csup\u003e2+\u003c/sup\u003e inside and outside the membrane is mainly done by Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase, and Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase (Jilka et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Hogaboom and Fedan., 1980; Weber et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e-ATPase can catalyze ATP hydrolysis on the inner side of the plasma membrane, release energy, and drive intracellular calcium ions to pump out of cells or pump them into the lumen of the endoplasmic reticulum for storage, so as to maintain a low concentration of free Ca\u003csup\u003e2+\u003c/sup\u003e in cells (Choi, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Because its activity depends on the combination of ATP and Mg\u003csup\u003e2+\u003c/sup\u003e, it forms a calcium pump together with Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase, which is an important basis for maintaining low Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis in nerve cells (Weber et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase is also an ion pump, providing energy for the active transport of Mg\u003csup\u003e2+\u003c/sup\u003eagainst the concentration gradient, thereby maintaining a relatively constant Mg\u003csup\u003e2+\u003c/sup\u003e concentration and osmotic pressure inside and outside the cell membrane(Tomita et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tashiro et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase and Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase activities in the CGs of earthworms by cypermethrin will lead to abnormal distribution of Ca\u003csup\u003e2+\u003c/sup\u003e inside and outside the nerve membrane, causing intracellular calcium overload. At the same time, the decrease of Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase activity may cause the decline of intracellular Mg\u003csup\u003e2+\u003c/sup\u003e, weaken the physiological antagonism to the increase of Ca\u003csup\u003e2+\u003c/sup\u003e, and aggravate calcium overload.\u003c/p\u003e \u003cp\u003eSince Ca\u003csup\u003e2+\u003c/sup\u003e plays an important role in the induction and maintenance of hippocampal long-term potentiation (LTP), a synaptic model of cognition and memory function (Smith, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Miyamoto and Fukunaga., 1996; Devine et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the abnormal Ca\u003csup\u003e2+\u003c/sup\u003e distribution inside and outside the neural membrane and intracellular calcium overload will affect the cognition and memory behavior of earthworms. In this study, the cognition and memory disabilities in earthworms were observed only at 6 mg/kg cypermethrin-treated groups, Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase, and Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase activities in earthworms exposure to 6 mg/kg cypermethrin were inhibited compared with controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but there was no difference in these ATPase activities between the control and 2 mg/kg cypermethrin (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that the intracellular calcium overload caused by inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase and Mg\u003csup\u003e2+\u003c/sup\u003e-ATPase activities may be one of the mechanisms of cognition and memory impairment in earthworms induced by cypermethrin exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Binding model of cypermethrin and Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase\u003c/h2\u003e \u003cp\u003eGiven that the abnormal Ca\u003csup\u003e2+\u003c/sup\u003e distribution inside and outside the nerve membrane of earthworms triggered by cypermethrin exposure may be a possible mechanism leading to neurobehavioral changes such as cognitive deficits in earthworms, and Ca\u003csup\u003e2+\u003c/sup\u003e-ATP is one of the main enzymes determining the Ca\u003csup\u003e2+\u003c/sup\u003e distribution inside and outside the nerve membrane of earthworms, the Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase was chosen as the receptor for molecular docking to explore the molecular mechanism of the neurotoxicity of cypermethrin in earthworms (Colina et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Qi et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe three-dimensional structure of the Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase receptor of the earthworm needs to be obtained using homology modeling. A PDB sequence number 5ZTF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.), which is known and has the highest homology to the Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase primary sequence, was selected as a template for homology modeling. The homology similarity between the study subject \u003cem\u003eAmynthas corticis\u003c/em\u003e Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase (GWHGAOSM004429.1) and 5ZTF was 71.59%. The Ramachandran plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.) shows that the percentage of GWHGAOSM004429.1 protease residues falling into the most favorable region is 95.49%, while the percentage of residues located in the region not in the active site are all less than 4.51%, indicating that the structure of the Ca\u003csup\u003e2+\u003c/sup\u003e-ATP protein model modeled by using the 5ZTF as a template homology is in general reasonable and can be used for molecular docking.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of molecular docking of GWHGAOSM004429.1 with cypermethrin are shown in Fig.\u0026nbsp;6. Residues LYS-728 and ALA-725 in GWHGAOSM004429.1 interacted with cypermethrin to form two hydrogen bonds with lengths of 2.0 and 2.7A, respectively. The binding energy of GWHGAOSM004429.1 with cypermethrin was \u0026minus;\u0026thinsp;8.17 kcal/mol, suggesting that cypermethrin interacts with Ca\u003csup\u003e2+\u003c/sup\u003e-ATP receptor proteins and that this interaction occurs spontaneously and a lower energy and more stable complex is obtained, the formation of which consumes Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase, resulting in a decrease in Ca\u003csup\u003e2+\u003c/sup\u003e -ATPase content and a decrease in enzyme activity. In this study, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB showed that cypermethrin exposure inhibited Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase activity within earthworm CGs, which is consistent with the results of the molecular docking of cypermethrin and Ca\u003csup\u003e2+\u003c/sup\u003e-ATP receptor proteins.\u003c/p\u003e \u003cp\u003eThe results of molecular docking of cypermethrin with Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase showed that cypermethrin inhibited earthworm Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase activity by interacting with Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase to form a stable complex. With the formation of this complex, the Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase content in the CGs of earthworms continued to decrease, which triggered the abnormal distribution of Ca\u003csup\u003e2+\u003c/sup\u003e inside and outside the nerve membranes of earthworms, and the intracellular calcium overload, which led to neurobehavioral changes, such as cognition and memory deficits in earthworms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Comparison of subchronic neurotoxicity using the integrated biomarker response index\u003c/h2\u003e \u003cp\u003eThe IBR method is a comprehensive evaluation tool to assess integrated toxicity effects in an organism(Hou et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study, responses from six biomarkers were normalized and expressed as star plots using exposure values for the 2, 6 mg/kg cypermethrin amendments on 28 d and 56 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e.). Since the IBR was the sum of normalized values for all indicators, the larger the normalized value, the bigger the influence of pesticides on earthworm (Normalized value greater than 0 means activated, less than 0 means suppressed). In addition, the IBR values for cypermethrin are also presented in bar chart format (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e.) with larger values indicating greater neurotoxicity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e., the IBR values for 2, 6 mg/kg cypermethrin were 1.46, and 4.78 at 28 d, respectively, indicating that the neurotoxicity of cypermethrin to earthworms depended on the exposure concentration, the more exposure concentration, the greater of neurotoxicity of cypermethrin to earthworms at 28 d. With increased exposure time, the IBR values for 2, 6 mg/kg cypermethrin were 2.61, and 7.04 at 56 d, respectively, the IBR value for cypermethrin at 56 d was greater than that of the same concentration at 28 d, indicating that the subchronic neurotoxicity of cypermethrin to earthworms increased with increasing exposure time.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe results of the subchronic neurotoxicity study of earthworms exposed to cypermethrin showed that although the chronic neurotoxicity of cypermethrin did not lead to the death of earthworms, it induced neurobehavioral changes, such as locomotor retardation and cognitive deficits in earthworms. The inhibition of Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase activity in earthworms triggered by the interaction of cypermethrin with Ca\u003csup\u003e2+\u003c/sup\u003e-ATPase to form a stable complex is one of the possible molecular mechanisms of cognitive impairment in earthworms.\u003c/p\u003e \u003cp\u003eThe subchronic neurotoxicity of cypermethrin for earthworms increased with the increase of the exposure concentration and the duration of cypermethrin exposure, and the subchronic neurotoxicity of cypermethrin to earthworms leads to neurological damage in earthworms, but whether this neurological damage recovers with the elimination of the exposure remains to be further investigated.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science Foundations of China (No. 31860155), the Yunnan Natural Sciences Foundations (No.2017FG001(-040)), and the Natural Sciences Foundations of Key Laboratory of State Forestry Administration on Highly-Efficient Utilization of Forestry Biomass Resources in Southwest China (No. 2022-KF09).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable (this article does not contain studies involving human participants or their tissues)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot \u0026nbsp;applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFuHui Zhao\u003c/strong\u003e: Conceptualization, Formal analysis, Investigation, Methodology, Data curation, Writing-original draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSijia Wu\u003c/strong\u003e:\u0026nbsp;Conceptualization, Visualization, Resources, Formal analysis, Investigation, Writing-original draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShiPing Zhou\u003c/strong\u003e: Conceptualization, Validation, Writing-review \u0026amp; editing, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuiJuan Li\u003c/strong\u003e: Investigation, Formal analysis, Resources.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQiSheng Li\u003c/strong\u003e: Investigation, Formal analysis, Resources.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShouQing Liu\u003c/strong\u003e: Investigation, Methodology, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuaYin Liu:\u0026nbsp;\u003c/strong\u003eSoftware, Formal analysis, Resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMei Qin\u003c/strong\u003e: Formal analysis, Data Curation, Supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbramson, C.I., Buckbee, D.A., 1995. 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Saf. 244, 114038.\u003c/li\u003e\n\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":"Earthworms Cypermethrin Neurobehavioral Neurotoxicity Molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-4097539/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4097539/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCypermethrin is one of the most heavily used pyrethroid pesticides worldwide and is a potential threat to soil organisms such as earthworms. In this paper, \u003cem\u003eAmynthas corticis\u003c/em\u003e was selected as a test organism to investigate the neurobehavioral changes in movement, cognition and memory caused by subchronic neurotoxicity of cypermethrin in earthworms, starting from the changes in the characteristic enzymes of earthworms' nerve ion channels triggered by exposure to cypermethrin. The changes in biomarkers of earthworms were evaluated using the integrated biomarker response (IBR), and the mechanism of cypermethrin neurotoxicity in earthworms was investigated using molecular docking technology, so as to investigate the subchronic neurotoxicity of earthworms caused by exposure to cypermethrin. The results showed that the subchronic neurotoxicity of cypermethrin for earthworms increased with the increase of the exposure concentration and the duration of cypermethrin exposure. The chronic neurotoxicity of cypermethrin did not lead to earthworm death but induced neurobehavioral changes such as locomotor retardation and cognitive deficits in earthworms. Cypermethrin exposure induced abnormalities in the enzyme that characterizes nerve ion channels in earthworms, which is one of the possible molecular mechanisms for the neurobehavioral changes of locomotor retardation and cognition and memory disorders in earthworms.\u003c/p\u003e","manuscriptTitle":"Subchronic Neurotoxic Effects of Cypermethrin on Earthworms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-17 15:28:18","doi":"10.21203/rs.3.rs-4097539/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":"ee493b8e-752b-4403-99b8-6a4000a77ec2","owner":[],"postedDate":"April 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-01T03:57:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-17 15:28:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4097539","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4097539","identity":"rs-4097539","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}