A method for judging the influence of the environment on the development degree of chironomid larvae

Abstract

The present invention discloses a method for judging the influence of the environment on the development degree of chironomid larvae, including: setting a standard value for the area of the imaginal disc of chironomid larvae; calculating the experimental value of the area of the imaginal disc of at least one chironomid larva sample; and characterizing the development of the larvae based on the comparison between the experimental value of the area of the imaginal disc and the standard value of the area of the imaginal disc. The present invention characterizes the development of larvae based on the maximum area of the imaginal disc sections of chironomid larvae, and has the advantages of simple operation and short time consumption, and can further improve the indexes of ecological toxicology experiments with chironomids as model organisms.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic toxicology, and specifically relates to a method for judging the influence of the environment on the development degree of chironomid larvae and its application. Background Art

[0002] Chironomidae (non-biting midges), belonging to the suborder Nematocera of the order Diptera in the class Insecta, chironomids have strong adaptability and are almost distributed in all freshwater environments with different bottom sediments and water qualities. Their larvae are generally benthic insects in freshwater bodies, and the biomass accounts for 70%-80% of the total benthic organisms. Both chironomid larvae and adults are food sources for higher trophic level organisms. Therefore, chironomids occupy an important ecological position in the ecosystem. Due to their high sensitivity to pollutants and short life cycles, chironomid larvae are often used for environmental indication and toxicological evaluation and have become typical test organisms. As a holometabolous insect, chironomids need to go through four stages in their life cycle: egg, larva, pupa, and adult. Their first to fourth instar larvae live in the sediments at the bottom of the water body. This special living habit makes chironomids an ideal material for evaluating the effects of chemical drugs on aquatic organisms. Microscopic tissue changes in organisms are important histological indicators for studying the effects of exogenous pollutants on the growth and development of organisms and have been widely used in environmental biology. Chironomids are considered representative organisms for the ecological toxicity test of freshwater benthic invertebrates because of their wide distribution, easy feeding, and strong reproductive ability under laboratory conditions. However, due to the small size of chironomids, there are great difficulties in preparation and dissection, and there are relatively few reports on the application of histological indicators related to chironomids in ecotoxicology. Imaginal discs are unique structures in the larval stage of insects composed of undifferentiated cells, and will form structures such as the wings and legs of adults as the insects develop continuously. As a marker of the developmental stage of insects, their development is of decisive significance for the smooth pupation and emergence of insects.

[0003] The existing indicators for observing and judging the influence of the environment on chironomids include mortality, reproduction rate, etc. These are all characterized by the direct consequences of the environment on chironomids and cannot reflect the specific influence of the environment on the development and tissue growth of chironomid larvae, that is, the prior art cannot accurately characterize the growth and development of chironomid larvae. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for judging the influence of the environment on the development degree of chironomid larvae, which can characterize the growth and development of chironomid larvae by the size of the imaginal disc area.

[0005] A method for judging the influence of the environment on the development degree of chironomid larvae includes:

[0006] S101. Set the standard value of the imaginal disc area of chironomid larvae;

[0007] Obtain paraffin sections of samples of at least one chironomid larva in a normal developmental state, determine the maximum area of the imaginal disc of the chironomid larva, and then use the maximum area of the imaginal disc of the chironomid larva as the standard value of the imaginal disc area of the chironomid larva;

[0008] Among them, the method for obtaining paraffin sections of samples of at least one chironomid larva in a normal developmental state and determining the maximum area of the imaginal disc of the chironomid larva includes (1)-(3);

[0009] (1) Sample collection: Take multiple chironomids, continuously culture the chironomids in the laboratory for 10 generations, then culture the chironomids after 10 generations of culture in dechlorinated fresh water aerated for 48 h, maintain the temperature at 23±2 °C, the light-dark ratio at 16:8, and the light intensity at 800±200 lux; Feed artificial feed during the feeding period, feed three times a week at 1.0 mg / individual / time until multiple chironomid egg strings within 8 h after new production are obtained, separately culture the chironomid egg strings until the early fourth instar and sample them as the chironomid larvae for setting the standard value of the imaginal disc area of the chironomid larva; Place the chironomid larvae in clean water for exposure; When the chironomid larvae develop to the late fourth instar, collect them in the clean water using a 10 mL plastic pipette to obtain several chironomid larva samples;

[0010] (2) Sample preparation: Among the several chironomid larva samples, fix the surviving and unbroken chironomid larvae in 4% paraformaldehyde to obtain multiple fixed chironomid larvae, and continuously cross-section the second to fourth body segments of the fixed chironomid larvae at intervals of 10 μm and perform HE staining to obtain chironomid larva sample sections with the maximum cross-section of the imaginal disc;

[0011] (3) Area measurement: Observe the chironomid larva sample sections using a scientific-grade stereomicroscope, determine the position of the imaginal disc in the fourth instar chironomid larvae, select the chironomid larva sample section with the maximum area of the imaginal disc of the chironomid larva, and determine the maximum area of the imaginal disc of the chironomid larva;

[0012] Among them, the method for determining the maximum area of the imaginal disc of the chironomid larva includes S1011-S1013:

[0013] S1011. Obtain at least one set of chironomid larva sample sections containing the imaginal disc, and the at least one set of chironomid larva sample sections containing the imaginal disc are derived from the at least one chironomid larva; The imaginal disc is located on both sides of the center of the chironomid larva sample section;

[0014] S1012. Screen out the first maximum value from the first imaginal disc areas of at least one set located on one side of the center of the chironomid larva sample section and screen out the second maximum value from the second imaginal disc areas of at least one set located on the other side of the center of the chironomid larva sample section;

[0015] S1013. After summing the first maximum value and the second maximum value, divide the sum by twice the number of chironomid larvae to obtain the maximum area of the adult disk of the chironomid larvae.

[0016] S102. Calculate the experimental value of the adult disk area of at least one chironomid larva sample in a preset environment, including:

[0017] Obtain paraffin sections of at least one chironomid larva sample affected by the preset environment, determine the maximum area of the adult disk of the chironomid larva, and then use the maximum area of the adult disk of the chironomid larva as the experimental value of the adult disk area of the chironomid larva.

[0018] S103. Based on the comparison between the experimental value of the adult disk area and the standard value of the adult disk area, determine whether the chironomid larvae are affected by the preset environment; when the difference between the experimental value of the adult disk area and the standard value of the adult disk area is greater than the standard threshold, it is determined that the chironomid larvae are affected by the promotion of the preset environment and cause overdevelopment; when the difference between the experimental value of the adult disk area and the standard value of the adult disk area is less than the standard threshold, it is determined that the chironomid larvae are affected by the inhibition of the preset environment and cause underdevelopment; otherwise, it is determined that the chironomid larvae are not affected by the preset environment.

[0019] Among them, the method for determining the standard threshold includes:

[0020] Using the standard value data of the adult disk area of chironomid larvae, calculate the standard threshold based on the least significant difference method.

[0021] The standard threshold is calculated according to the following formula:

[0022]

[0023] Among them: is the two-sided α / k quantile of the t-distribution with degrees of freedom N - k, where N is the total number of observations of the paraffin sections of chironomid larvae, k is the number of chironomid larvae, and each chironomid larva contains a set of paraffin section samples of chironomid larvae, and α is the significance level (such as 0.05); MSE is the mean squared error, which is obtained from the ANOVA table in existing data; n is the number of paraffin sections in each chironomid larva (assuming that the number of paraffin sections in all groups is the same).

[0024] Note: Through the above method, the tissue development of chironomid larvae can be characterized by the size of the area value of the adult disk, and the impact of the environment on chironomid larvae can be reflected in gene expression and biological tissues, thus achieving a technical effect that cannot be characterized by existing technical indicators.

[0025] Further, the method for setting the standard value of the imaginal disc area of the chironomid larvae includes:

[0026] Obtain paraffin sections of at least one sample of chironomid larvae in a normal developmental state, determine the maximum area of the imaginal disc of the chironomid larvae, and then use the maximum area of the imaginal disc of the chironomid larvae as the standard value of the imaginal disc area of the chironomid larvae.

[0027] Note: The above process can obtain the imaginal disc area of chironomid larvae without being affected by specific conditions as the standard value for subsequent judgment.

[0028] The method for calculating the experimental value of the imaginal disc area of at least one sample of chironomid larvae in a preset environment includes:

[0029] Obtain paraffin sections of at least one sample of chironomid larvae affected by the preset environment, determine the maximum area of the imaginal disc of the chironomid larvae, and then use the maximum area of the imaginal disc of the chironomid larvae as the experimental value of the imaginal disc area of the chironomid larvae.

[0030] Note: The above method can select the most representative imaginal disc development sample section from the chironomid larvae sample sections of each group of imaginal discs, which can better highlight the developmental differences of chironomid larvae. That is to say, by selecting the imaginal disc with the largest area for comparison, it can not only accurately reflect the developmental situation but also highlight the differences between each group of samples, making the calculation results more intuitive and thus improving the accuracy of judgment.

[0031] Further, the method for determining the standard threshold includes:

[0032] Use the data of the standard value of the imaginal disc area of chironomid larvae, and calculate the LSD critical value based on the least significant difference method, and use the LSD critical value as the standard threshold.

[0033] Note: The above method can determine whether there is a significant difference in the experimental value of chironomid larvae by the data of the standard value of the imaginal disc area of chironomid larvae and the difference from the experimental value, and then obtain the degree of influence of the environment on chironomid larvae.

[0034] Further, when no imaginal disc is observed on one side of the center of the chironomid larvae sample section and an imaginal disc is observed on the other side of the center of the chironomid larvae sample section, set the area of the imaginal disc on one side of the center of the chironomid larvae sample section to 0 and participate in the calculation of the experimental value of the imaginal disc area.

[0035] Note: The above reason may be that the experimental chironomid larvae are affected by the test substance, the imaginal disc develops abnormally and is missing on one side. To accurately reflect this situation, it needs to be included in the statistics at this time.

[0036] Further, when imaginal discs are not observed on both sides of the center of the mosquito larva sample section, it indicates that this mosquito larva sample section is not usable.

[0037] Explanation: During experimental operations such as embedding, dehydration, and cutting, the specific body segments of the larva may be deformed due to extrusion or shaking, and the imaginal discs may experience a certain displacement. In this case, it is possible that the imaginal discs on one or both sides cannot be observed, and at this time, observation cannot be carried out, that is, the section is not usable.

[0038] Further, the preset environment includes an aquatic environment or an aquatic sediment environment, and the aquatic environment or the aquatic sediment environment contains bisphenol compounds.

[0039] Taking the maximum average area of the imaginal discs of the above-mentioned control group (i.e., the standard value of the imaginal disc area of Chironomus larvae) as a reference, comparing the maximum average area of the imaginal discs in the treatment group (the experimental value of the imaginal disc area of the Chironomus larva sample), using statistical methods to calculate the significance and characterizing the effect of the test drug on the development of Chironomus larvae with specific data; specifically:

[0040] If the maximum area of the imaginal discs in the treatment group is significantly smaller than the maximum area of the imaginal discs in the control group (i.e., exceeding the standard threshold), it indicates that the development of the larvae in the treatment group is inhibited compared to the control group;

[0041] If the maximum area of the imaginal discs in the treatment group is significantly larger than the maximum area of the imaginal discs in the control group, it indicates that the development of the larvae in the treatment group is promoted compared to the control group;

[0042] If there is no significant difference in the maximum area of the imaginal discs in the treatment group compared to the maximum area of the imaginal discs in the control group, and there is also no significant difference in aspects such as the appearance shape, it indicates that there is no apparent effect on the development of the imaginal discs of the larvae in the treatment group compared to the control group.

[0043] The beneficial effects of the present invention are:

[0044] The present invention can characterize the tissue development of Chironomus larvae by using the size of the area value of the imaginal discs, and can reflect the influence of the environment on Chironomus larvae in terms of gene expression and biological tissues, thereby achieving a technical effect that cannot be characterized by existing technical indicators. It has the advantages of simple operation and short time consumption, and can further improve the indicators of the ecotoxicology experiment with Chironomus as a model organism. Description of the Drawings

[0045] Figure 1 It is a data graph of the acute toxicity effect of BPF on Chironomus tentans larvae in the embodiment of the present invention;

[0046] Figure 2It is the graph of the developmental toxicity data of BPF on chironomid larvae in the embodiments of the present invention;

[0047] Figure 3 It is a schematic diagram of the section of the effect of BPF on the development of chironomid imaginal discs in the embodiments of the present invention;

[0048] Figure 4 It is the graph of the data of the effect of BPF exposure on the oviposition rate of different genders of Chironomus tentans in the embodiments of the present invention. Detailed implementation manners

[0049] To further elaborate on the methods adopted and the effects achieved by the present invention, the technical solutions of the present invention will be clearly and completely described below in combination with experiments.

[0050] In water bodies or ecological environments, there are various chemical substances that have an impact or harm on organisms. In the process of characterizing the impact of these chemical substances on organisms, conventional methods such as the mortality rate of organisms and the statistics of death events, the pupation rate and the statistics of pupation time are usually used. These methods are all in the form of existing results and cannot characterize the changes in the developmental state and internal mechanism of organisms when they are affected. The following are the existing indicators of the impact of chemical substances in the existing environment on chironomid larvae:

[0051] Acute toxicity mortality rate:

[0052] Judgment criterion: If the larvae do not show obvious displacement within 15 s after gently impacting the larvae with a pipette to suck water, it is regarded as being inhibited.

[0053] Experimental operation: Add a certain amount of the test substance to water and set a control group. Add first-instar larvae to the test beakers, expose the first-instar chironomid larvae to a concentration range of the test substance, and observe their death or inhibition conditions at 24 h and 48 h. These data can be used to calculate the median effective concentration EC50, etc. through a regression model, or the NOEC / LOEC can be determined through statistical hypothesis testing. The concentration range of the test should cover the effective concentration (such as EC 15 , EC 50 ) and the effective concentration that receives special attention. Generally speaking, when the effective concentration is within the test concentration range, it can improve the accuracy, especially the effectiveness, of the prediction of the effective concentration (ECx). Extrapolation much lower than the lowest positive concentration or higher than the highest concentration should be avoided. A preliminary test for finding the range helps in the selection of the test range. If ECx is to be predicted, at least 5 concentrations should be set in the test, with 3 replicates for each concentration. In any case, it is advisable to set sufficient test concentrations for good model prediction. If the number of test concentrations showing different responses is increased, the number of replicates for each treatment can be reduced. Increasing the number of replicates or reducing the size of the test concentration interval will result in narrowing the confidence interval of the test.

[0054] formula:

[0055] Chronic toxicity:

[0056] The concentration design was based on the acute toxicity data results. In this experiment, the fourth-instar larvae were selected to carry out a 240-h chronic toxicity test to observe their mortality, pupation rate and pupation time.

[0057] Overall experimental operation: Before the experiment, take out the newly laid egg clusters within 8 hours and culture them separately until the early fourth instar to sample the larvae for the experiment. The larvae are randomly added to the treatment group and the control group for exposure. The experimental concentration is set based on the acute toxicity experiment, and the concentration close to EC10, EC20, and EC50 is selected. Each group has 3 parallels, and each parallel has 5 larvae.

[0058] mortality rate:

[0059] Judgment criteria: After using a straw to suck water and gently impact the larvae, the dead larvae will be motionless and stiff, or disappear, or have a mutilated body and abnormal white color, which is considered dead.

[0060] Experimental operation: Observe the death of larvae in the beaker every 24 hours, remove dead larvae in time, and count the number of deaths and experimental time.

[0061] formula:

[0062] Pupation rate:

[0063] Judgment criteria: The larvae of Chironomus tentans are generally cylindrical and worm-shaped, with the body length of the last instar larvae ranging from 10 to 15 mm. The newly hatched larvae are translucent white as a whole, with a body length of only about 700 μm. As time goes by, the body color gradually turns red, and the body length and weight also increase significantly. The head of the larva is oval in dorsal view, and sensory organs such as ocelli and antennae, as well as lips, chins, jaws, etc. are all concentrated on the front side of the head. The body is divided into 12 body segments in total. The outer surface of each body segment is smooth without long slender setae. The first 3 body segments are thoracic segments, and the last 9 body segments are abdominal segments. The thoracic segments and abdominal segments are almost of the same width, without obvious swelling of the thoracic segments. A pair of procercopods grow on the first thoracic segment, and fine cilia grow at the ends of the procercopods, which can be used to collect fine particles to build nests. Two pairs of ventral tubes grow on both sides of the 8th abdominal segment for excretion. A pair of metacercopods is present at the end of the 9th abdominal segment. The larval stage of Chironomus tentans lasts for 18 - 20 days, and the body length and weight increase with the increase of the development time. During this period, it needs to go through 3 molts. The growth rate of the body length and weight will increase short-term after each molt. Thus, the larval stage can be divided into 4 instars. The newly hatched instar I larvae will secrete mucus from the body surface to bond the collected fine particles to form a tubular nest, and the nest will expand continuously with the growth and development of the larvae. At the end of the IV instar, the thoracic segments of the larvae begin to swell and fuse. The remaining 9 body segments are abdominal segments, and the length also gradually shortens. Finally, it molts to form a pupa with a length of only 5 - 7 mm. The head and thorax of the pupa are fused into the cephalothorax. Fine white root-like villi grow on both sides of the front end, which are the respiratory organs of the pupal stage. The rudiments of wings and legs have already developed. The width of the abdomen is slightly smaller than that of the cephalothorax. The body color of the pupa will deepen continuously with time. The pupal stage is the shortest stage, only lasting for 1 - 2 days; Determine the experimental time and the total number of larvae pupating. It is necessary to count every day during the expected pupation period. Record the number of fully pupated individuals every day.

[0064] Experimental operation: Observe the pupation situation of the larvae in the beaker every 24 hours, and remove the pupae in time, and count the number of successfully pupated individuals and the experimental time.

[0065] Formula:

[0066] Pupation start time:

[0067] Experimental operation: Count the pupation time of the first successfully pupated larva in each parallel under each concentration group

[0068] Formula:

[0069]

[0070] Pupation end time:

[0071] Experimental operation: Count the pupation time of the last successfully pupated larva in each parallel under each concentration group Formula:

[0072]

[0073] Among the above-mentioned indicators, they are all result-oriented intuitively to characterize the impact of the environment on chironomid larvae. However, in some slightly toxic or complex environments, the chemical substances in the environment do not directly harm chironomid larvae, but will affect and interfere at the tissue and gene levels, which may affect the growth and development of the offspring of chironomid larvae. Therefore, considering the ecological safety of the environment, an indicator that can characterize the tissue and development of chironomid larvae is needed to analyze the impact of the environment on chironomid larvae.

[0074] Combined with the above content, the following embodiments of the present invention set new evaluation indicators for chironomid larvae to more accurately reflect the development and other conditions of chironomid larvae.

[0075] Example 1: A method for judging the influence of the environment on the development degree of chironomid larvae, including:

[0076] S101. Set the standard value of the area of the imaginal disc of chironomid larvae;

[0077] Specifically: Obtain paraffin sections of samples of at least one chironomid larva in a normal development state, determine the maximum area of the imaginal disc of the chironomid larva, and then use the maximum area of the imaginal disc of the chironomid larva as the standard value of the area of the imaginal disc of the chironomid larva.

[0078] Exemplarily, the paraffin sections of samples of chironomid larvae in a normal development state are recorded as the control group and the following treatments (1) to (3) are carried out.

[0079] (1) Sample collection: Continuously culture chironomids in the laboratory for 10 generations. The chironomids are cultured in dechlorinated fresh water with aeration for more than 48 hours, and gentle aeration is maintained in the pre-culture tank, with a temperature of 23 ± 2°C, a light-dark ratio of 16:8, and a light intensity of 800 ± 200 lux. During the feeding period, artificial feed is fed, and they are fed three times a week at 1.0 mg / individual / time. Before the experiment, the egg strings within 8 hours after new production are taken out and cultured separately until the early fourth instar, and the larvae sampled are used as the experimental larvae. The chironomid larvae are placed in the control group in clean water (i.e., chironomid larvae in a normal development state) for exposure. When the larvae in the control group develop to the late fourth instar, the larval samples of the control group and the treatment group are collected on the same day. When collecting the larval samples, a 10 mL plastic pipette is used, and the action of sucking the larvae is required to be gentle and rapid to reduce the stimulation to the larvae and reduce the mortality of the larvae during the sample collection process;

[0080] (2) Sample preparation: Among the above-mentioned control group (i.e., chironomid larvae in normal developmental state), select the surviving larvae whose bodies are not damaged during collection, and immediately fix the samples in 4% paraformaldehyde. Perform serial paraffin sectioning and HE staining on the second to fourth body segments (transverse section) of the larvae. Cut 10 consecutive sections (at 10 μm intervals) for each sample to observe the effect of a certain drug on larval development. The purpose of serial sectioning is to cut the largest cross-section of the imaginal disc. The more sections are cut and the smaller the interval, the more likely it is to observe the largest area of the imaginal disc. Select paraffin sections with intact tissues and clear staining to further measure the largest area of the cross-section of the imaginal disc of chironomid larvae (measure the imaginal discs of at least five larvae in each group).

[0081] (3) Area determination: Observe the serial sections using a scientific-grade stereomicroscope (Nikon SMZ25) and take pictures. Determine the position of the imaginal disc in the fourth instar chironomid larvae based on all the obtained paraffin section images. Select the section with the largest area of the imaginal disc in each group and measure the largest area of the imaginal disc in each group. The purpose of serial paraffin sectioning and taking pictures is to screen out the pictures of the imaginal disc with the largest area. Arrange and number the previous sections in the order of the cutting process. For a group of ten consecutive sections of each larva at each concentration, when observing the imaginal disc of each group of sections, it is necessary to find that the area of the imaginal disc follows an order of increasing, then decreasing, and then increasing again. The section with the largest area of the imaginal disc is the largest area of the imaginal disc of this larva. At this time, use a microscope with an area measurement function for measurement. Since the imaginal discs are distributed on both sides of the insect body, when measuring, calculate based on the two sets of data obtained by measuring each side of one larva separately, and there is no need to add them. Note that the largest area of the imaginal discs on both sides of the insect body may exist in different sections during sectioning. In this case, measure the largest area of the corresponding side of the two sections.

[0082] The method for determining the largest area of the imaginal disc of chironomid larvae includes S1011 - S1013:

[0083] S1011. Obtain at least one set of paraffin sections of chironomid larva samples containing imaginal discs, and the at least one set of paraffin sections of chironomid larva samples containing imaginal discs is derived from the at least one chironomid larva; the imaginal discs are located on both sides of the center of the paraffin sections of the chironomid larva samples.

[0084] S1012. Screen out the first maximum value from at least one set of first imaginal disc areas on one side of the center of the paraffin sections of the chironomid larva samples and screen out the second maximum value from at least one set of second imaginal disc areas on the other side of the center of the paraffin sections of the chironomid larva samples.

[0085] S1013. After summing the first maximum value and the second maximum value, divide the sum by twice the number of chironomid larvae to obtain the largest area of the imaginal disc of the chironomid larvae.

[0086] S102. Calculate the experimental value of the imaginal disc area of at least one chironomid larva sample in a preset environment, including:

[0087] Obtain paraffin sections of at least one chironomid larva sample affected by the preset environment, determine the maximum area of the imaginal disc of the chironomid larva, and then use the maximum area of the imaginal disc of the chironomid larva as the experimental value of the imaginal disc area of the chironomid larva.

[0088] Exemplarily, bisphenol F is selected to simulate a toxic environment for the test. After the test, the chironomid larvae are used to prepare samples according to (1) to (3) in the above step S101 and obtain the experimental value of the imaginal disc area; the specific process and results are shown in Experimental Example 1 and Experimental Example 2 below.

[0089] S103. Based on the comparison between the experimental value of the imaginal disc area and the standard value of the imaginal disc area, determine whether the chironomid larva is affected by the preset environment; when the difference between the experimental value of the imaginal disc area and the standard value of the imaginal disc area is greater than the standard threshold, it is determined that the chironomid larva is affected by the promoting effect of the preset environment and causes overdevelopment; when the difference between the experimental value of the imaginal disc area and the standard value of the imaginal disc area is less than the standard threshold, it is determined that the chironomid larva is affected by the inhibitory effect of the preset environment and causes underdevelopment; otherwise, it is determined that the chironomid larva is not affected by the preset environment; wherein, the preset environment includes a water environment or a water sediment environment, and the water environment or the water sediment environment contains bisphenol compounds.

[0090] Further, when no imaginal disc is observed on one side of the center of the chironomid larva sample section and an imaginal disc is observed on the other side of the center of the chironomid larva sample section, set the area of the imaginal disc on one side of the center of the chironomid larva sample section to 0 and participate in the calculation of the experimental value of the imaginal disc area. When no imaginal disc is observed on both sides of the center of the chironomid larva sample section, it means that this chironomid larva sample section is unusable.

[0091] It should be understood that the principle of the above judgment is: when the imaginal disc of an insect only exists on one side and the organizational structure cannot be found on the other side in consecutive paraffin sections, there are the following possibilities:

[0092] 1. The experimental chironomid larvae are affected by the test substance, the imaginal disc develops abnormally, and one side is missing. At this time, calculate according to the area of the imaginal disc on one side being 0μm 2 and it cannot be not counted;

[0093] 2. There is an operation error during the paraffin sectioning process, and this section needs to be discarded in this case;

[0094] 3. During experimental operations such as embedding, dehydration, and cutting, the specific body segments of the larvae were deformed due to extrusion or shaking, and the imaginal discs may have undergone a certain displacement, which has a probability of causing one or both imaginal discs to be unobservable;

[0095] 4. The range where the imaginal discs exist is from the 2nd to the 4th body segments of the larvae. This range is only the data obtained from a large number of experiments on chironomid larvae in our laboratory. There may be slight differences between different species, and it needs to be determined according to the organisms cultured in the laboratory and the experiments;

[0096] 5. The larval instar was too small, resulting in the developmental situation of the imaginal discs being unobservable. In this case, the chironomid larvae selected were at the end of the fourth instar, and the developmental situation of the imaginal discs in the control group was better. If the imaginal discs that cannot be observed are not due to the above reasons, then researchers need to analyze specifically according to the specific problems.

[0097] The method for determining the above standard threshold includes:

[0098] Using the standard value data of the area of the imaginal discs of chironomid larvae, the LSD critical value is calculated based on the Least Significant Difference method, and the LSD critical value is used as the standard threshold.

[0099] Specifically, the Least Significant Difference (LSD) method is a method used to compare the differences between the means of multiple samples, especially after analysis of variance (ANOVA). When the ANOVA result shows that there are significant differences between at least two groups. The LSD method calculates a critical value (i.e., the above standard threshold) to determine which group means have statistically significant differences.

[0100] Steps to determine the critical value (standard threshold):

[0101] Conduct ANOVA analysis: First, ANOVA analysis needs to be performed on all groups to test whether there are overall significant differences between them. If the p - value of ANOVA is less than the significance level (usually 0.05), it is considered that there are significant differences between at least two groups.

[0102] Calculate the LSD critical value: After ANOVA confirms the existence of significant differences, the LSD method can be used to further determine which group differences are significant; the LSD critical value is usually calculated based on the following formula:

[0103]

[0104] Where: is the two-sided α / k quantile of the t-distribution with N-k degrees of freedom, where N is the total number of observations, k is the number of groups, and α is the significance level (e.g., 0.05); MSE is the mean squared error, obtained from the ANOVA table;

[0105] n is the number of samples in each group (assuming the same number of samples in all groups).

[0106] That is to say, taking the maximum average area of the control group's imaginal discs (i.e., the standard value of the imaginal disc area of chironomid larvae) as a reference, comparing the maximum average area of the imaginal discs in the treatment group (the experimental value of the imaginal disc area of chironomid larva samples), statistical methods are used to calculate the significance and specific data are used to characterize the effect of the test drug on the development of chironomid larvae. Specifically:

[0107] If the maximum area of the imaginal discs in the treatment group is significantly smaller than that in the control group (i.e., exceeding the standard threshold), it indicates that the development of the larvae in the treatment group is inhibited compared to that in the control group;

[0108] If the maximum area of the imaginal discs in the treatment group is significantly larger than that in the control group, it indicates that the development of the larvae in the treatment group is promoted compared to that in the control group;

[0109] If there is no significant difference in the maximum area of the imaginal discs between the treatment group and the control group, and there is also no significant difference in aspects such as the appearance shape, it indicates that there is no apparent effect on the imaginal disc development of the larvae in the treatment group compared to those in the control group.

[0110] Experimental Example 1: Prove the feasibility of the embodiment of the present invention in judging the influence of the environment on chironomid larvae based on imaginal discs;

[0111] Conduct a developmental toxicity experiment of BPF (bisphenol F) on chironomids. The experimental chironomids (Chironomus tentans) were continuously cultured in the laboratory for more than 10 generations. Freshwater dechlorinated and aerated for more than 48 h was used for breeding, and gentle aeration was maintained in the pre-breeding tank at a temperature of 23±2°C, a light-dark ratio of 16:8, and a light intensity of 800±200 lux. During the feeding period, artificial feed (Qingdao Longxing FEED Co, Ltd.) was fed, and the worms were fed three times a week at 1.0 mg / worm / time. The BPF used in this study (purity >99.5%, purchased from Solarbio) was dissolved in DMSO as a stock solution before the experiment, and different doses were added according to the concentration.

[0112] A total of 8 BPF concentrations (0.1, 0.5, 1.0, 2.0, 4.0, 8.0, 10.0, 20.0 mg / L) were set for the acute toxicity experiment, with 4 parallel groups for each concentration; there were 5 first-instar larvae in each parallel group. The exposure period was 48 h, and the larvae were not fed or aerated during the experiment. The inhibition of larvae at 24 h and 48 h was recorded, and the acute activity inhibition rate of the larvae was calculated. If the larvae did not show obvious displacement within 15 s after gently flushing them with a pipette to suck water, they were considered inhibited;

[0113] Before the chronic toxicity test, egg strings produced within 8 h were taken out and cultured separately until the early fourth instar, and then sampled as the larvae for the experiment. The larvae were randomly added to the prepared 50.0 mL BPF experimental solution and the control group without bisphenol substances (recorded as Control) for exposure. Based on the acute toxicity experiment, the experimental concentrations were set, and concentrations close to EC 10 、EC 20 、EC 50 were selected (1.5, 2.0, 2.5, 3.0, 4.0 mg / L). Each group had 3 parallels, with 5 larvae in each parallel. During the experiment, the larvae were fed at a ratio of 1.0 mg / larva / day, and gentle aeration was maintained during the experiment. The survival and pupation of the larvae in each group were counted every 24 h, and the experiment continued until all the larvae pupated or died. Dead larvae were removed every day during the experiment. Additional parallel groups were added in the control group and the 2.0 and 4.0 mg / L treatment groups to collect samples for histopathological analysis of the larval imaginal discs.

[0114] As Figure 1 shown, compared with the control group, the acute activity inhibition of the larvae by BPF at each concentration at 24 h and 48 h was as follows: after the larvae were exposed to 4.0 mg / L BPF for 24 h, the inhibition rate reached 20%, and increased to 45% after 48 h, significantly inhibiting the activity of the larvae (P<0.05); when exposed for 24 h or more, all the tested larvae above 8.0 mg / L were significantly inhibited, and the inhibition rate reached more than 95% (P<0.05). The inhibition rate of the larvae was positively correlated with the BPF exposure time and exposure concentration; the effective concentration was positively correlated with the inhibition rate and negatively correlated with the exposure time (as shown in Table 1).

[0115] Table 1 EC 10 ,EC 20 ,and EC 20

[0116]

[0117]

[0118] As Figure 2As shown, during the exposure period, all the larvae in the control group survived and developed normally to the pupation stage. The larval mortality rate in the BPF treatment group was positively correlated with the exposure concentration. When the BPF concentration was ≤ 1.5 mg / L, there was no significant difference in the larval mortality rate compared to the control group (P > 0.05). When the BPF concentration was ≥ 2.0 mg / L, the larval mortality rate was significantly higher than that of the control group (P < 0.05) (as shown in Figure 2 A in

[0119] The distribution of the pupation time and the cumulative pupation rate of the larvae under BPF exposure are shown in Figure 2B. Compared with the control group, the presence of BPF caused a lag in larval pupation; the cumulative pupation rate decreased with the increase in concentration. When the BPF concentration was ≤ 1.5 mg / L, there was a decreasing trend in the cumulative pupation rate, although it was not significant (P > 0.05). When the BPF concentration was ≥ 2.0 mg / L, the cumulative pupation rate of the larvae in all treatment groups decreased significantly (P < 0.05) (as shown in Figure 2 B in

[0120] The effects of BPF on the pupation time of the larvae and the time occupied by each stage are shown in Figure 2 C and E in

[0121] To explore the potential effects of BPF exposure on the development of larvae, referring to the identification of some organs and tissues of Aedes aegypti, the positions of the imaginal discs (green marks) in the fourth instar larvae of Chironomus tentans were determined. Under consecutive paraffin sections, Figure 3 E, J, and O in Figure 3 correspond to the largest areas of the imaginal discs of the fourth instar larvae in the control group, the 2.0 mg / L treatment group, and the 4.0 mg / L treatment group, respectively. The development degree of the imaginal discs was quantified by measuring the areas of the imaginal discs in consecutive sections (as shown in

[0122] As shown in B in

[0123] Compared with the control group, the area of the imaginal discs in the 4.0 mg / L BPF treatment group was significantly smaller than that of the control group (P < 0.05). The inhibitory effect of 4.0 mg / L BPF on the development of the larval imaginal discs was more obvious than that of the 2.0 mg / L group. In summary, in the embodiment of the present invention, taking the largest area of the imaginal discs of Chironomus tentans larvae as one of the indicators, the effects of BPF on the growth and development of Chironomus tentans larvae were confirmed. The results showed that this method was time-consuming, simple to operate, and convenient and fast for sample processing, and it was confirmed that 4.0 mg / L BPF could cause a significant inhibitory effect on the development of the larval imaginal discs.Furthermore, for the tissues and development of certain chironomid larvae, the above-mentioned indicators may not accurately characterize them, as shown in the following experiment:

[0124] Experimental Example 2: Prove the superiority of the embodiment of the present invention in judging the influence of the environment on chironomid larvae based on imaginal discs;

[0125] Reproductive toxicity: A total of three exposure concentrations were set. The highest concentration was set with reference to the lowest effective concentration in the developmental toxicity test, which were 0.5, 1.0, 2.0 mg / L treatment groups and a 0 mg / L control group. Each group had 3 replicates, and each replicate had 20 fourth-instar larvae. The experimental environmental conditions were the same as those in 2.2(2). During the exposure period, the emergence of extended chironomid larvae in the control group and the 3 treatment groups was continuously observed. At 08:00 and 20:00 every day, the newly emerged adults in each experimental group were collected twice and transferred to new 5L glass beakers filled with aerated fresh water according to male and female respectively. Healthy male and female adults were transferred in according to the ratio of female:male = 1:1 respectively for measuring the reproductive capacity of poisoned female insects and poisoned male insects, and the oviposition rate of each group was recorded.

[0126] Calculation method of oviposition rate: It was calculated based on the number of egg strings laid by each female mosquito. The oviposition rate value was the total number of egg strings produced in each experimental container divided by the total number of female mosquitoes that survived and had no damage in the experimental container. In the control group and the treatment groups, additional parallel groups were added to synchronously measure the expression levels of genes related to ecdysteroid hormone, juvenile hormone, and insulin pathways in adults less than 12 h old after eclosion.

[0127] Experimental results:

[0128] As Figure 4 shown, the influence of BPF on the oviposition rate of chironomids (at the individual level - has no influence on the oviposition rate of female adults)

[0129] The influence of BPF with reproductive toxicity on the oviposition rate of chironomids under different treatments is shown in Fig 5A - B. Compared with the control group, there was no significant difference in the oviposition rate of poisoned female chironomids × unpoisoned male chironomids in the four BPF concentration treatment groups, which was between 85% - 95% (P > 0.05). For the oviposition rate of poisoned male chironomids × unpoisoned female chironomids, the oviposition rate of male mosquitoes in the 0.5 mg / L treatment group was 93%, which had no significant difference compared with the control group (P > 0.05); the oviposition rates of the 1.0 mg / L and 2.0 mg / L treatment groups were 75% and 61% respectively, both significantly lower than the control group (P < 0.01).

[0130] In summary, the above-mentioned reproduction experiment characterized the effects of BPF on adult female chironomids. The experiment proved that no significant differences were found in terms of the reproduction indicators, that is, BPF had no significant effect on the reproduction of chironomids. However, for the adult disc area in the embodiments of the present invention, there was a significant difference compared with the control group, indicating that some genes were significantly up-regulated at the gene level. It is proved that using the adult disc area to characterize the development situation can observe phenomena that cannot be characterized by other indicators at the tissue level, and thus can accurately reflect the development and growth of chironomid larvae.

Claims

1. A method for judging the influence of the environment on the development degree of chironomid larvae, characterized in that, Including: S101. Set the standard value of the imaginal disc area of chironomid larvae; Obtain paraffin sections of at least one sample of chironomid larvae in a normal developmental state, determine the maximum area of the imaginal disc of the chironomid larvae, and then use the maximum area of the imaginal disc of the chironomid larvae as the standard value of the imaginal disc area of the chironomid larvae; wherein, the method for obtaining paraffin sections of at least one sample of chironomid larvae in a normal developmental state and determining the maximum area of the imaginal disc of the chironomid larvae includes (1)-(3); (1) Sample collection: Take multiple chironomids, continuously culture the chironomids in the laboratory for 10 generations, then culture the chironomids after 10 generations of culture in dechlorinated fresh water with aeration for 48 h, keep the temperature at 23±2°C, the light-dark ratio at 16:8, and the light intensity at 800±200 lux; feed artificial feed during the feeding period, feed three times per week at 1.0 mg / individual / time until multiple chironomid egg strings within 8 h after new production are obtained, separately culture the chironomid egg strings until the early fourth instar and sample them as the chironomid larvae for setting the standard value of the imaginal disc area of the chironomid larvae; place the chironomid larvae in clean water for exposure; when the chironomid larvae develop to the late fourth instar, collect them in the clean water using a 10 mL plastic pipette to obtain several chironomid larva samples; (2) Sample preparation: Among the several chironomid larva samples, fix the surviving and unbroken chironomid larvae in 4% paraformaldehyde to obtain multiple fixed chironomid larvae, continuously cross-section the second to fourth body segments of the fixed chironomid larvae at intervals of 10 μm and perform HE staining to obtain chironomid larva sample sections with the maximum cross-section of the imaginal disc; (3) Area measurement: Observe the chironomid larva sample sections using a scientific-grade stereomicroscope, determine the position of the imaginal disc in the fourth instar chironomid larvae, select the chironomid larva sample section with the maximum area of the imaginal disc of the chironomid larvae, and determine the maximum area of the imaginal disc of the chironomid larvae; Among them, the method for determining the maximum area of the imaginal disc of the chironomid larvae includes S1011-S1013: S1011. Obtain at least one group of chironomid larva sample sections containing the imaginal disc, and the at least one group of chironomid larva sample sections containing the imaginal disc are derived from the at least one chironomid larva; the imaginal disc is located on both sides of the center of the chironomid larva sample section; S1012. Screen out the first maximum value from the first imaginal disc areas of at least one group located on one side of the center of the chironomid larva sample section and screen out the second maximum value from the second imaginal disc areas of at least one group located on the other side of the center of the chironomid larva sample section; S1013. After summing the first maximum value and the second maximum value, divide the sum by twice the number of chironomid larvae to obtain the maximum area of the imaginal disc of the chironomid larvae; S102. Calculate the experimental value of the imaginal disc area of at least one chironomid larva sample in a preset environment, including: Obtain paraffin sections of at least one chironomid larva sample affected by the preset environment, determine the maximum area of the imaginal disc of the chironomid larvae, and then use the maximum area of the imaginal disc of the chironomid larvae as the experimental value of the imaginal disc area of the chironomid larvae; S103. Based on the comparison between the experimental value of the imaginal disc area and the standard value of the imaginal disc area, determine whether the chironomid larvae are affected by the preset environment; when the difference between the experimental value of the imaginal disc area and the standard value of the imaginal disc area is greater than the standard threshold, it is determined that the chironomid larvae are affected by the promotion of the preset environment and cause overdevelopment; when the difference between the experimental value of the imaginal disc area and the standard value of the imaginal disc area is less than the standard threshold, it is determined that the chironomid larvae are affected by the inhibition of the preset environment and cause underdevelopment; otherwise, it is determined that the chironomid larvae are not affected by the preset environment.

2. The method for judging the influence of the environment on the development degree of chironomid larvae according to claim 1, wherein The preset environment includes a water environment or a water sediment environment, and the water environment or the water sediment environment contains bisphenol compounds.

3. The method for judging the influence of the environment on the development degree of chironomid larvae according to claim 1, wherein When no imaginal disc is observed on one side of the center of the chironomid larva sample section and an imaginal disc is observed on the other side of the center of the chironomid larva sample section, set the area of the imaginal disc on one side of the center of the chironomid larva sample section to 0 and participate in the calculation of the experimental value of the imaginal disc area.

4. The method for judging the influence of the environment on the development degree of chironomid larvae according to claim 1, wherein The determination method of the standard threshold includes: Using the standard value data of the imaginal disc area of chironomid larvae, calculate the LSD critical value based on the least significant difference method, and use the LSD critical value as the standard threshold.

5. A method for judging the influence of the environment on the development degree of chironomid larvae according to claim 4, characterized in that, The calculation formula of the LSD critical value is as follows: In the formula, LSD is the LSD critical value: It is the two-sided α / k quantile of the t-distribution with N - k degrees of freedom, where N is the total number of observations of the paraffin sections of chironomid larvae, k is the number of chironomid larvae, and each chironomid larva contains a set of chironomid larva sample sections, and α is the significance level; MSE is the mean square error; n is the number of paraffin sections in each chironomid larva.

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