Method for evaluating the neurotoxic effect of heat stress on eriocheir sinensis and application thereof

Abstract

The application discloses a method for evaluating the influence of heat stress on the neurotoxicity of Eriocheir sinensis and application thereof, wherein the neurotoxicity refers to neuron heat damage toxicity caused by heat stress, the neuron apoptosis, DNA damage, neuron cell damage biomarker (NSE) and heat shock response of Eriocheir sinensis are used as judgment parameters of the neurotoxicity, and the above features are comprehensively analyzed to evaluate the heat damage toxicity of heat stress on the nerve tissue of Eriocheir sinensis. The evaluation method has high reliability, operability and practicability, provides a new method for accurately detecting and evaluating the neurotoxicity of Eriocheir sinensis, and provides help for screening Eriocheir sinensis fry groups with high high-temperature adaptability, high high-temperature tolerance and high survival ability.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method for evaluating the neurotoxic effects of heat stress on the Chinese mitten crab and its application. Background Technology

[0002] The Chinese mitten crab (scientific name: Eriocheir sinensis), commonly known as the river crab or mitten crab, is rich in nutrients and has a delicious taste. As one of the special aquatic products, it has a broad market demand.

[0003] With global climate change, the frequency and intensity of extreme heat events are constantly increasing. In this environment of global warming, organisms will inevitably face the threat of heat stress. Unlike terrestrial animals, aquatic animals are poikilothermic, meaning their body temperature changes with environmental temperature. Heat stress damage caused by elevated temperatures involves a series of disorders at the molecular, biochemical, and physiological levels. When fluctuations in environmental temperature exceed the heat threshold of aquatic animals, they may suffer fatal heat stress injuries. The central nervous system (CNS) is the control center for all life activities in an animal and is particularly sensitive to changes in environmental temperature. Heat stress is considered a key factor causing neuronal damage. Compared to other tissues and organs, the CNS is more susceptible to the effects of heat stress and the resulting heat-induced toxicity.

[0004] The Chinese mitten crab (Eriocheir sinensis) exhibits a narrow thermophilicity, with an optimal water temperature range of approximately 22-28℃. In recent years, unprecedented extreme heat events have occurred in major mitten crab farming areas in North my country, East China, and Central China. During summer, the water temperature in mitten crab farms can reach above 32℃, with some ponds even experiencing prolonged periods above 35℃. Therefore, sustained extreme heat can cause severe heat stress damage to mitten crabs. However, there is currently no method for evaluating the neurotoxic effects of heat stress on mitten crabs. For individuals, the nervous system is far more sensitive to heat stress than other tissues or organs, meaning that neurotoxicity will be a crucial indicator of an individual's heat tolerance. Therefore, providing a scientific, systematic, reliable, operable, and practical method for evaluating neurotoxicity is an urgent technical problem to be solved and is essential for the breeding of new heat-resistant varieties. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, this invention provides a method for evaluating the effects of heat stress on the neurotoxicity of Chinese mitten crab, providing a new method for accurately detecting and evaluating the neurotoxicity of Chinese mitten crab.

[0006] Technical Solution: To achieve the above objectives, the present invention provides a method for evaluating the neurotoxicity of heat stress on the Chinese mitten crab, comprising the following steps:

[0007] Step 1: Heat stress experiments were conducted using juvenile Chinese mitten crabs, with two groups receiving temperature treatments at 24℃ and 32℃ respectively;

[0008] Step 2: Set different sampling time points for the two groups with equal gradients to obtain the time of toxicity effect with the best sensitivity and significant effect;

[0009] Step 3: Use multidimensional sensitive parameters to evaluate neurotoxicity using a comprehensive approach;

[0010] The neurotoxicity refers to the thermal damage toxicity of neurons caused by heat stress induced by high temperatures.

[0011] In step one, juvenile crabs weighing 4.32±0.36g were randomly selected and randomly divided into two groups: a 24℃ suitable temperature group and a 32℃ high temperature group. A controllable water temperature heating system was used to keep the water temperature constant.

[0012] In step two, the two groups designed in step one are kept in the same culture conditions except for the difference in water temperature. After treatment for 0-96 hours, samples are taken and ganglia are collected as evaluation tissues to obtain the time of toxicity effect with the best sensitivity and significant effect.

[0013] As a preferred option, in step two, samples are taken after processing at 0h, 12h, 24h, 48h, and 96h respectively.

[0014] In step two, the toxicity effect time is obtained by comprehensively analyzing the heat shock response and apoptosis gene expression at different time points.

[0015] In step three, after selecting the optimal time point, a comprehensive evaluation of neurotoxicity is conducted using multidimensional sensitive parameters, including pathological phenotype, apoptosis gene expression, neuronal cell damage biomarkers, and heat shock response.

[0016] Furthermore, the pathological phenotypes described in step three are the visualization analysis of neuronal apoptosis and neuronal DNA damage; the apoptosis gene expression is the expression level of cysteine-containing aspartate proteolytic enzyme genes (Casepase 3 and Casepase 8); the neuronal cell damage biomarker is the content of neuron-specific enolase (NSE) in serum; and the heat shock response is the expression level of heat shock protein genes (HSP 90 and HSP 70).

[0017] In step three, the neurotoxicity and its degree are evaluated by comprehensively analyzing the visualization and significance of parameter differences between differential groups.

[0018] The method described in this invention for evaluating the effects of heat stress on the neurotoxicity of Chinese mitten crab is used to accurately detect and evaluate the neurotoxicity of Chinese mitten crab.

[0019] The method for evaluating the effects of heat stress on the neurotoxicity of Chinese mitten crab described in this invention is applied in screening out Chinese mitten crab seedling populations that have strong high-temperature adaptability, high-temperature tolerance, and strong survival ability.

[0020] The application of a reagent or tool for visual analysis of neuronal apoptosis, neuronal DNA damage, serum neuron-specific enolase (NSE) levels, and heat shock protein expression levels in Chinese mitten crabs in evaluating the neurotoxic effects of heat stress on Chinese mitten crabs.

[0021] The neurotoxicity described in this invention refers to the toxicity of heat damage to neurons caused by heat stress. This invention uses neuronal apoptosis, DNA damage, and neuronal cell damage biomarkers (NSE) of the Chinese mitten crab as parameters for assessing neurotoxicity. A comprehensive analysis of these characteristics is conducted to evaluate the toxicity of heat stress on the nerve tissue of the Chinese mitten crab. The evaluation method of this invention is highly reliable, operable, and practical, providing a new method for accurately detecting and evaluating the neurotoxicity of the Chinese mitten crab.

[0022] Existing technologies reveal that the nervous system is highly sensitive to high temperatures. Sustained high temperatures pose a threat of heat stress to animal bodies, and this heat stress causes the most severe heat damage and toxicity to the nervous system. Compared to mammals, aquatic poikilothermic animals have extremely poor thermoregulation capabilities and are more susceptible to the effects of heat stress. Currently, there is no research on the effects of heat stress on the nervous system of aquatic animals, nor is there a method for evaluating neurotoxicity. For individual animals, the nervous system is far more sensitive to heat stress than other organs, meaning that neurotoxicity will be a crucial indicator of their heat tolerance. Therefore, this invention proposes for the first time a scientific and accurate method for evaluating neurotoxicity, which is essential for the breeding of new heat-resistant varieties.

[0023] This invention presents for the first time a scientific, systematic, reliable, operable, and practical method for evaluating the neurotoxicity of the Chinese mitten crab.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0025] This invention provides and applies a method for evaluating the neurotoxicity of Chinese mitten crabs, using neuronal apoptosis and DNA damage as visualized pathological phenotypes, NSE content as a biomarker of neuronal damage, and heat shock response as a heat stress-sensitive parameter. The evaluation method of this invention is highly reliable, operable, and practical, providing a new approach for accurately detecting and evaluating the neurotoxicity of Chinese mitten crabs.

[0026] The method of this invention can screen for populations of Chinese mitten crabs with strong high-temperature adaptability, which is of great significance for the breeding of new high-temperature resistant varieties. Attached Figure Description

[0027] Figure 1 The time-dependent effects of heat stress on heat shock response and apoptosis gene expression in the ganglia of *Eriocheir sinensis* (Note: *P<0.05; **P<0.01; ***P<0.001);

[0028] Figure 2 Visual analysis of the effects of heat stress on neuronal apoptosis in Chinese mitten crab;

[0029] Figure 3 The results of a comet experiment on neurons of the Chinese mitten crab under heat stress;

[0030] Figure 4 The effect of heat stress on the NSE content in the serum of Chinese mitten crab (Note: ***P<0.001). Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0032] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0033] Example 1

[0034] This embodiment describes a method for evaluating the effects of heat stress on the neurotoxicity of the Chinese mitten crab and its application.

[0035] Juvenile Chinese mitten crabs were purchased from a commercial aquaculture farm in Chongming, Shanghai. After two weeks of temporary rearing, juvenile crabs with good vitality, uniform size, and intact limbs (weighing 4.32 ± 0.36 g) were selected and randomly assigned to two groups: a suitable temperature group and a high-temperature group. The suitable temperature group was maintained at 24℃, and the high-temperature group at 32℃. A controllable water temperature heating system was used to control and maintain a constant water temperature, and all groups were raised under the same routine conditions.

[0036] Samples were collected at 0h, 12h, 24h, 48h, and 96h after stress treatment. Thoracic ganglia were collected as evaluation tissues. The expression levels of heat shock protein genes (HSP 90 and HSP 70) and apoptosis genes (Casepase 3 and Casepase 8) were detected by qRT-PCR to obtain the optimal time for toxicity effect with the best sensitivity and significant effect. The results are as follows: Figure 1As shown, heat stress can induce heat shock and apoptosis in the nervous system within 12 hours, with significant effects and a time effect. The toxicity was most pronounced at 96 hours, and 96 hours was chosen for further investigation.

[0037] The specific implementation methods for gene expression levels are as follows:

[0038] Gene expression levels were determined according to the kit instructions. Total RNA was extracted from neural tissue using RNAiso™ plus reagent (RNA extraction kit, Vazyme Biotech Co., Ltd., Nanjing, China). The concentration and mass of total RNA were estimated using a Nano Drop 2000 spectrophotometer (Thermo, USA) and analyzed using PrimeScript according to the kit instructions. TM Reverse transcription was performed using an RT kit (Perfect Real Time, TaKaRa, Japan). Sample quality was standardized to an A260 / 280 ratio of 1.8 to 2.0. ChamQ was used. TM Universal Relative quantification and analysis were performed using the qPCR Master Mix kit (Vazyme Biotech Co., Ltd., Nanjing, China) and the CFX96 Real-Time PCR system (Bio-rad, Richmond, CA). The program was as follows: 94℃, 3 min; 40 cycles, 94℃, 1 sec. β-actin and S27 were used as internal controls. Primers are shown in Table 1. Relative changes in gene expression levels were analyzed using 2... -ΔΔCt The method has been determined.

[0039] Table 1 Primer Information

[0040]

[0041] Furthermore, using 96 hours as the optimal time point for toxicity effect with the best sensitivity and significant effect, a multidimensional sensitivity parameter was employed to evaluate neurotoxicity using a comprehensive approach.

[0042] Pathological phenotypes: Visual analysis of neuronal apoptosis and neuronal DNA damage;

[0043] Neuronal cell damage biomarker: NSE level;

[0044] The specific implementation method for visual analysis of neuronal apoptosis is as follows:

[0045] The FITC TUNEL assay was used, which mainly includes a series of steps such as fixation, dehydration and paraffin embedding of tissues (brain and thoracic ganglia of juvenile Chinese mitten crab), sectioning, dewaxing and rehydration, room temperature equilibration, TUNEL reaction, DAPI nuclear counterstaining, and mounting. Specific methods were followed according to the literature (Neural excitotoxicity and the toxic mechanism induced by acute hypoxia in Chinese mitten crab (Eriocheir sinensis)[J]. Aquatic Toxicology, 2022, 245: 106131.). Paraffin sections were 4 μm thick. Microscopic examination and image acquisition were performed using an ortho-fluorescent microscope (Eclipse Ci-L, Nikon Corporation, Japan). A PANNORAMIC panoramic slide scanner (3DHISTECH, Hungary) was used to scan tissue sections and collect imaging information. Results are as follows: Figure 2 As shown, high-temperature-induced heat stress caused significant apoptosis of neurons in both the cerebral and thoracic ganglia of the Chinese mitten crab.

[0046] The specific implementation method for evaluating neuronal DNA damage using comet assays is as follows:

[0047] The main steps include cell suspension preparation, gel coating, cell lysis, DNA alkaline unwinding, single-cell electrophoresis, neutralization, and staining. Specific methods are detailed in the instructions for the comet electrophoresis method of the DNA damage kit (G010-1-1, Nanjing Jiancheng Bioengineering Institute). The cell density of the cell suspension from the ganglia of juvenile Chinese mitten crabs was 1×10⁻⁶ cells / mL. 6 Single-cell electrophoresis was performed at 25V for 20–30 min. Voltage and current could be adjusted by changing the buffer level. After electrophoresis, the slides were placed in a petri dish. 0.4 mmol / L Tris-HCl (pH 7.5) buffer was added, immersing the slides completely. The mixture was neutralized three times at 4°C for 10 min each time. The Tris-HCl buffer was discarded, and 20 μl of PI staining solution was added to each slide. The slides were then covered with coverslips and stained in the dark for 10 min. After staining, the slides were examined and photographed using a fluorescence microscope. The results are as follows: Figure 3 As shown, heat stress leads to the production of more fragments of neuronal DNA in the brain and thoracic ganglia of the Chinese mitten crab, forming a long trail, indicating that high-temperature induced heat stress causes severe damage to the neuronal DNA of the Chinese mitten crab.

[0048] The specific implementation method for determining the level of NSE, a biomarker of nerve cell damage, is as follows:

[0049] The level of NSE was determined using a crab neuron-specific enolase (NSE) enzyme-linked immunosorbent assay kit (MM-927504O1, Jiangsu Enzyme Immunosorbent Assay Co., Ltd.) employing a double-antibody sandwich method. Both NSE detection and extraction were performed using the kit method. Purified crab NSE antibody was coated onto microplates to prepare a solid-phase antibody. NSE was added sequentially to the wells coated with the monoclonal antibody, followed by binding with HRP-labeled NSE antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted to blue under the catalysis of HRP enzyme, and then to yellow under acidic conditions. The color intensity was positively correlated with the NSE concentration in the sample. The absorbance was measured at 450 nm using a microplate reader, and the NSE concentration in the sample was calculated using a standard curve. The results are shown below. Figure 4 As shown, high-temperature-induced heat stress significantly increased NSE levels in the serum of Chinese mitten crabs. NSE is a nerve cell-specific enzyme and a sensitive indicator and biomarker for assessing the severity of nerve cell damage. A significant increase in serum NSE levels indicates severe neuronal damage.

[0050] In summary, high-temperature induced heat stress significantly increased visual apoptosis in the neurons of *Eriocheir sinensis*, as evidenced by the consistent trend in the expression of apoptosis-related genes and heat shock protein genes. Furthermore, comet assays showed that high-temperature induced heat stress caused severe neuronal DNA damage. The level of the neuronal cell damage biomarker NSE was also significantly elevated in the serum of heat-stressed *Eriocheir sinensis*, confirming that heat stress caused neuronal damage. By combining these multidimensional parameters, the neurotoxic effects of heat stress on *Eriocheir sinensis* can be effectively evaluated, providing assistance in screening for *Eriocheir sinensis* seedlings with strong high-temperature adaptability, high heat tolerance, and high survival rate.

Claims

1. A method for evaluating the effects of heat stress on the neurotoxicity of the Chinese mitten crab, characterized in that, Includes the following steps: Step 1: Heat stress experiments were conducted using juvenile Chinese mitten crabs, with two groups receiving temperature treatments at 24℃ and 32℃ respectively; Step 2: Set different sampling time points for the two groups with equal gradients to obtain the time of toxicity effect with the best sensitivity and significant effect; Step 3: Use multidimensional sensitive parameters to evaluate neurotoxicity using a comprehensive approach; The neurotoxicity refers to the neuronal thermal damage toxicity caused by heat stress induced by high temperature. In step three, after selecting the optimal time point, a comprehensive evaluation of neurotoxicity is conducted using multidimensional sensitive parameters, including pathological phenotype, apoptosis gene expression, neuronal cell damage biomarkers, and heat shock response. The pathological phenotypes are the visualization analysis of neuronal apoptosis and neuronal DNA damage; the apoptosis gene expression is the expression level of the cysteine-containing aspartate proteolytic enzyme gene; the neuronal cell damage biomarker is the content of neuron-specific enolase (NSE) in serum; and the heat shock response is the expression level of the heat shock protein gene.

2. The method for evaluating the effects of heat stress on the neurotoxicity of *Eriocheir sinensis* according to claim 1, characterized in that, In step one, juvenile crabs weighing 4.32 ± 0.36g were randomly selected and randomly divided into two groups: a 24℃ suitable temperature group and a 32℃ high temperature group. A controllable water temperature heating system was used to keep the water temperature constant.

3. The method for evaluating the effects of heat stress on the neurotoxicity of *Eriocheir sinensis* according to claim 1, characterized in that, In step two, the two groups designed in step one were kept in the same culture conditions except for the difference in water temperature. After treatment for 0-96 hours, samples were taken and ganglia were collected as evaluation tissues to obtain the time of toxicity effect with the best sensitivity and significant effect.

4. The method for evaluating the effects of heat stress on the neurotoxicity of *Eriocheir sinensis* according to claim 3, characterized in that, In step two, samples are taken after processing at 0h, 12h, 24h, 48h, and 96h respectively.

5. The method for evaluating the effects of heat stress on the neurotoxicity of *Eriocheir sinensis* according to claim 3, characterized in that, In step two, the heat shock response and apoptosis gene expression at different time points are comprehensively analyzed to obtain the time of toxicity effect with the best sensitivity and significant effect.

6. The method for evaluating the effects of heat stress on the neurotoxicity of *Eriocheir sinensis* according to claim 1, characterized in that, In step three, the visualization and significance of parameter differences between differential groups are comprehensively analyzed to evaluate neurotoxicity and its degree.

7. The method for evaluating the effects of heat stress on the neurotoxicity of Chinese mitten crab as described in claim 1 is used for the accurate detection and evaluation of the neurotoxicity of Chinese mitten crab.

8. The application of the method for evaluating the effects of heat stress on the neurotoxicity of Chinese mitten crab as described in claim 1 in screening out Chinese mitten crab seedling populations with strong high-temperature adaptability, high-temperature tolerance, and strong survival ability.

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