Method for judging influence of environment on development degree of chironomidae larvae

Abstract

The invention discloses a method for judging the influence of the environment on the development degree of chironomidae larvae. The method comprises the following steps: setting an adult disc area standard value of the chironomidae larvae; calculating an adult disc area experimental value of at least one chironomidae larva sample; on the basis of comparison between the adult tray area experiment value and the adult tray area standard value, the development condition of the insects is represented. The larva development condition is represented based on the maximum area of the chironomidae larva adult plate slice, the method has the advantages of being easy to operate, short in consumed time and the like, and the indexes of ecotoxicology experiments with chironomidae as model organisms can be further improved.

Description

Technical Field

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

[0002] Chironomidae (non-biting midges) belongs to the family Chironomidae of the order Diptera and the suborder Longicornis of the class Insecta. Chironomidae have strong adaptability and are found in almost all freshwater environments with different bottom and water quality conditions. Their larvae are generally benthic insects in freshwater bodies, and their biomass accounts for 70%-80% of the total benthic organisms. Both chironomid larvae and adults are food sources for organisms at higher trophic levels, so chironomids occupy an important ecological position in the ecosystem. Due to their high sensitivity to pollutants and short life cycle, chironomid larvae are often used in environmental indicators and toxicological assessments and have become typical test organisms. As a holometabolous insect, chironomids need to go through four stages in their life: eggs, larvae, pupae, and adults. 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 chemicals on aquatic organisms. Changes in the microscopic structure of 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 organisms. Chironomids are considered representative organisms for ecotoxicity testing of freshwater benthic invertebrates because of their wide distribution, ease of feeding, and strong reproductive capacity under laboratory conditions. However, due to the small size of Chironomids, there are great difficulties in preparation and dissection. There are relatively few reports on the application of histological indicators related to Chironomids in ecotoxicology. The imaginal disc is a structure unique to the larval stage of insects composed of undifferentiated cells. As the insect continues to develop, it will form structures such as wings and legs of the adult. As a sign of the developmental stage of the insect, its development is of decisive significance for the successful pupation and emergence of the insect.

[0003] Existing indicators for observing and judging the impact of the environment on chironomids include mortality rate, reproduction rate, etc. These all characterize the direct consequences of the environment on chironomids and cannot reflect the specific impact of the environment on the development and tissue growth of chironomid larvae. In other words, existing technologies cannot accurately characterize the growth and development of chironomid larvae. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for judging the influence of 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 determining the effect of environment on the development of chironomid larvae, comprising:

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

[0007] Calculate the experimental value of the imaginal disc area of ​​at least one chironomid larvae sample in a preset environment;

[0008] Based on the comparison between the experimental value of the imaginal disc area and the standard value of the imaginal disc area, it is judged whether the chironomid larvae are affected by the preset environment; when the difference between the standard value of the imaginal disc area and the standard value of the imaginal disc area is greater than the standard threshold value, it is judged that the chironomid larvae are promoted by the preset environment and cause overdevelopment; when the difference between the standard value of the imaginal disc area and the standard value of the imaginal disc area is less than the standard threshold value, it is judged that the chironomid larvae are inhibited by the preset environment and cause underdevelopment; otherwise, it is judged that the chironomid larvae are not affected by the preset environment.

[0009] Note: Through the above method, the area value of the imaginal disc can be used to characterize the tissue development of chironomid larvae, and the impact of the environment on chironomid larvae can be reflected in gene expression and biological tissue conditions, thereby achieving technical effects that cannot be characterized by existing technical indicators.

[0010] Furthermore, the method for setting the standard value of the imaginal disc area of ​​the chironomid larvae comprises:

[0011] Obtain a paraffin section of at least one sample of a 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.

[0012] Note: The above process can prepare the imaginal disc area of ​​the chironomid larvae when they are not affected by specific conditions, which can be used as a standard value to facilitate the subsequent judgment process.

[0013] The method for calculating the experimental value of the imaginal disc area of ​​at least one chironomid larvae sample in a preset environment comprises:

[0014] Obtain a paraffin section of at least one chironomid larvae sample after being affected by a preset environmental environment, determine the maximum area of ​​the chironomid larvae imaginal disc, and then use the maximum area of ​​the chironomid larvae imaginal disc as the experimental value of the chironomid larvae imaginal disc area.

[0015] Furthermore, the method for determining the maximum area of ​​the imaginal disc of the chironomid larvae comprises:

[0016] Obtain at least one group of chironomid larvae sample slices containing imaginal discs, wherein the at least one group of chironomid larvae sample slices containing imaginal discs are derived from at least one chironomid larva; the imaginal discs are located on both sides of the center of the chironomid larvae sample slices;

[0017] A first maximum value is selected from at least one group of first imaginal disc areas located on one side of the center of the chironomid larvae sample slice, and a second maximum value is selected from at least one group of second imaginal disc areas located on the other side of the center of the chironomid larvae sample slice;

[0018] The first maximum value and the second maximum value are summed and then divided by twice the number of the chironomid larvae to obtain the maximum area of ​​the chironomid larvae imaginal disk.

[0019] Note: The above method can select the most representative adult disc development sample slices from the chironomid larvae sample slices in each group of insect discs, and can better highlight the developmental differences of chironomid larvae. That is to say, selecting the adult disc with the largest area for comparison can not only accurately reflect the development situation, but also highlight the differences between each group of samples, making the calculation results more intuitive, thereby improving the accuracy of judgment.

[0020] Furthermore, the method for determining the standard threshold includes:

[0021] The LSD critical value was calculated based on the least significant difference method using the standard value data of the imaginal disc area of ​​chironomid larvae, and the LSD critical value was used as the standard threshold.

[0022] Note: The above method can be used to determine whether the experimental value of chironomid larvae has significant differences through the standard value data of the imaginal disc area of ​​chironomid larvae and the difference with the experimental value, and then to obtain the degree of influence of the environment on the chironomid larvae.

[0023] Furthermore, when no imaginal disk is observed on one side of the center of the chironomid larvae sample slice and an imaginal disk is observed on the other side of the center of the chironomid larvae sample slice, the area of ​​the imaginal disk on one side of the center of the chironomid larvae sample slice is set to 0 and participates in the calculation of the experimental value of the imaginal disk area.

[0024] Note: The above reason may be that the experimental chironomid larvae were affected by the test substance, and the imaginal disc was deformed and one side was missing. In order to accurately reflect this situation, it needs to be included in the statistics.

[0025] Furthermore, when no imaginal discs are observed on both sides of the center of the mosquito larvae sample slice, it indicates that the mosquito larvae sample slice is unusable.

[0026] Note: Because specific segments of the larvae are squeezed or shaken and deformed during experimental operations such as embedding, dehydration, and cutting, the imaginal discs may be displaced to a certain extent. This may result in one or both sides of the imaginal disc being unable to be observed. In this case, observation cannot be made, and the slices are unusable.

[0027] Furthermore, the preset environment includes a water environment or a water sediment environment, and the water environment or the water sediment environment contains bisphenol compounds.

[0028] A method for determining the effect of environment on the development of chironomid larvae, comprising:

[0029] S101, setting a standard value for the imaginal disc area of ​​chironomid larvae;

[0030] Obtain a paraffin section of at least one sample of a 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;

[0031] The method of obtaining a paraffin section of at least one chironomid larvae sample in a normal developmental state and determining the maximum area of ​​the imaginal disc of the chironomid larvae comprises (1) to (3);

[0032] (1) Sample collection: Take a plurality of chironomids, culture the chironomids in the laboratory for 10 generations, and then culture the chironomids after 10 generations in aerated dechlorinated fresh water for 48 hours, 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 breeding period, feed three times a week at 1.0 mg / bird / time, until a plurality of chironomid egg strings newly produced within 8 hours are obtained, and the chironomid egg strings are cultured separately to the early stage of the fourth instar and sampled 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 stage of the fourth instar, use a 10 mL plastic pipette to collect them in the clean water to obtain a plurality of chironomid larvae samples;

[0033] (2) Sample preparation: Among the plurality of chironomid larvae samples, the chironomid larvae that are alive and intact are fixed in 4% paraformaldehyde to obtain a plurality of fixed chironomid larvae, and the second to fourth body segments of the fixed chironomid larvae are continuously cross-sectioned 10 times at intervals of 10 μm and stained with HE to obtain a chironomid larvae sample section with the largest cross section of the imaginal disc;

[0034] (3) Area measurement: using a scientific research-grade stereo microscope to observe the chironomid larvae sample slices, determine the position of the imaginal disc in the fourth-instar chironomid larvae, select the chironomid larvae sample slices with the largest area of ​​the chironomid larvae imaginal disc, and determine the maximum area of ​​the chironomid larvae imaginal disc;

[0035] Wherein, the method for determining the maximum area of ​​the imaginal disc of the chironomid larvae comprises S1011-S1013:

[0036] S1011, obtaining at least one group of chironomid larvae sample slices containing imaginal discs, wherein the at least one group of chironomid larvae sample slices containing imaginal discs are derived from at least one chironomid larva; the imaginal discs are located on both sides of the center of the chironomid larvae sample slices;

[0037] S1012, selecting a first maximum value from at least one group of first imaginal disc areas located on one side of the center of the chironomid larvae sample slice, and selecting a second maximum value from at least one group of second imaginal disc areas located on the other side of the center of the chironomid larvae sample slice;

[0038] S1013, summing the first maximum value and the second maximum value, and dividing the sum by twice the number of the chironomid larvae to obtain the maximum area of ​​the chironomid larvae imaginal disc;

[0039] S102, calculating the experimental value of the imaginal disc area of ​​at least one chironomid larvae sample in a preset environment, including:

[0040] Obtaining a paraffin section of at least one chironomid larvae sample after being affected by a preset environmental environment, determining the maximum area of ​​the chironomid larvae imaginal disc, and then using the maximum area of ​​the chironomid larvae imaginal disc as an experimental value of the chironomid larvae imaginal disc area;

[0041] S103, based on the comparison between the experimental value of the imago disc area and the standard value of the imago disc area, judging whether the chironomid larvae are affected by the preset environment; when the difference between the standard value of the imago disc area and the standard value of the imago disc area is greater than the standard threshold, judging that the chironomid larvae are promoted by the preset environment and cause overdevelopment; when the difference between the standard value of the imago disc area and the standard value of the imago disc area is less than the standard threshold, judging that the chironomid larvae are inhibited by the preset environment and cause underdevelopment; otherwise, judging that the chironomid larvae are not affected by the preset environment;

[0042] The method for determining the standard threshold value includes:

[0043] The standard threshold is calculated based on the least significant difference method using the standard value data of the imaginal disc area of ​​the chironomid larvae; the standard threshold is calculated according to the following formula:

[0044]

[0045] Where: t α / k , N-k is the two-sided α / kQuantile, where N is the total number of observed paraffin sections of chironomid larvae, k is the number of chironomid larvae, each of which contains a set of chironomid larvae sample sections, α is the significance level (such as 0.05); MSE is the mean squared error, obtained from the existing data ANOVA table; n is the number of paraffin sections in each chironomid larvae (assuming that the number of paraffin sections in all groups is the same).

[0046] The maximum average area of ​​the imaginal disc of the control group (i.e., the standard value of the imaginal disc area of ​​the chironomid larvae) was used as a reference to compare the maximum average area of ​​the imaginal disc in the treatment group (the experimental value of the imaginal disc area of ​​the chironomid larvae sample), and the significance was calculated using statistical methods and specific data were used to characterize the effect of the test drug on the development of the chironomid larvae; specifically:

[0047] If the maximum area of ​​the insect disc of the treatment group is significantly smaller than the maximum area of ​​the imaginal disc of the control group (i.e., exceeds the standard threshold), it indicates that the development of the larvae in the treatment group is inhibited compared with that in the control group;

[0048] If the maximum area of ​​the insect disc of the treatment group is significantly larger than that of the adult disc of the control group, it means that the development of the larvae in the treatment group is promoted compared with that in the control group;

[0049] If there is no significant difference in the maximum area of ​​the imaginal disc of the treated group compared with the maximum area of ​​the imaginal disc of the control group, and there is no significant difference in appearance and shape, it means that there is no apparent effect on the development of the imaginal disc of the larvae in the treated group compared with the larvae in the control group.

[0050] The beneficial effects of the present invention are:

[0051] The present invention can characterize the tissue development of chironomid larvae by using the area value of the imaginal disc, and can reflect the influence of the environment on the chironomid larvae in terms of gene expression and biological tissue, thereby achieving a technical effect that cannot be characterized by the existing technical indicators. It has the advantages of simple operation and short time consumption, and can further improve the indicators of ecotoxicological experiments using chironomids as model organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is a data diagram of the acute toxic effect of BPF on Chironomus tentans larvae in an embodiment of the present invention;

[0053] Figure 2 This is a data graph of the developmental toxicity of BPF to chironomid larvae in an embodiment of the present invention;

[0054] Figure 3 Schematic diagram of the effect of BPF on the development of chironomid imaginal discs in an embodiment of the present invention;

[0055] Figure 4 This is a data graph showing the effect of exposure of Chironomus tentans of different sexes to BPF on the egg-laying rate in an example of the present invention. DETAILED DESCRIPTION

[0056] In order to further illustrate the method adopted by the present invention and the effect achieved, the technical solution of the present invention will be clearly and completely described in combination with experiments below.

[0057] In water bodies or ecological environments, there are a variety of 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 and death event statistics of organisms, pupation rate and pupation time statistics are usually used. These methods are in the form of existing results and cannot characterize the changes in the developmental state and internal mechanisms of organisms when they are affected. The following are existing indicators of the impact of chemical substances in the existing environment on chironomid larvae:

[0058] Acute toxicity mortality:

[0059] Judgment criteria: If the larvae do not move significantly within 15 seconds after being gently impacted with water using a pipette, they are considered to be inhibited.

[0060] Experimental operation: Add a certain amount of the test substance to the water and set up a control group. Add first-instar larvae to the test beaker, expose the first-instar chironomid larvae to a concentration range of the test substance, and observe their death or inhibition at 24h and 48h. These data can be used to calculate the half-maximum effect concentration EC50 through regression models, and can also be used to determine NOEC / LOEC through statistical hypothesis tests. The concentration range of the test should cover the effect concentration (e.g. EC 15 ,EC 50 ) and effect concentrations of particular interest. In general, the accuracy and especially the validity of effect concentration (ECx) predictions are improved when the effect concentration is within the experimental concentration range. Extrapolation too much below the lowest positive concentration or above the highest concentration should be avoided. Preliminary experiments to find the range can help in the selection of the experimental range. If ECx is to be predicted, at least 5 concentrations should be set in the experiment with 3 replicates each. In any case, it is wise to set enough experimental concentrations to obtain good model predictions. If the number of experimental concentrations that show 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 experimental concentration interval will result in a narrowing of the confidence interval of the experiment.

[0061] formula:

[0062] Chronic toxicity:

[0063] 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.

[0064] 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.

[0065] mortality rate:

[0066] 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.

[0067] 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.

[0068] formula:

[0069] Pupation rate:

[0070] Judgment criteria: The larvae of the extensor midge are generally cylindrical worm-like, and the body length of the last instar larvae varies from 10 to 15 mm. The newly hatched larvae are translucent white in color and only about 700 μm in length. As time goes by, the body color gradually turns red, and the body length and weight also increase significantly. The dorsal view of the larvae's head is oval, and the sensory organs such as eyespots and antennae, as well as the lips, chin, and jaws are all concentrated on the front side of the head. The body is divided into 12 segments, each with a smooth outer surface and no slender bristles. The first three segments are thoracic segments, and the last nine segments are abdominal segments. The thoracic segments are almost the same width as the abdominal segments, and there is no obvious thoracic swelling. The first thoracic segment grows a pair of anterior protopods, and the end of the anterior protopods grows fine cilia, which can be used to collect tiny particles for nesting. There are two pairs of abdominal tubes on the left and right sides of the 8th abdominal segment for excretion. There is a pair of posterior protopods at the end of the 9th abdominal segment. The larval stage of Chironomids extensa lasts for 18-20 days. The body length and weight increase with the increase of development time. During this period, it needs to undergo 3 molts. The body length and weight gain rate after each molt will increase for a short period of time. Therefore, the larval stage can be divided into 4 instars. The newly hatched I-instar larvae will use the mucus secreted from the body surface to glue the collected fine particles to create a tubular nest, and the nest will continue to expand as the larvae grow and develop. The thorax of the larvae at the end of the IV-instar begins to swell and heal, and the remaining 9 segments are abdominal segments, which gradually shorten in length. Finally, the larvae molt to form a pupa with a length of only 5-7mm. The head and thorax of the pupa are fused into the cephalothorax, and white root-like fine hairs grow on both sides of the front end, which are the respiratory organs of the pupa. The rudiments of wings and feet have developed. The width of the abdomen is slightly smaller than the cephalothorax, and the body color of the pupa will continue to deepen over time. The pupal stage is the shortest stage, which lasts only 1-2 days; the experimental time and the total number of pupated larvae are measured. It is necessary to count every day during the expected pupation period. The number of fully pupated pupae was recorded daily.

[0071] Experimental procedures: Observe the pupation of larvae in the beaker every 24 hours, remove the pupae in time, and count the number of successful pupations and the experimental time.

[0072] formula:

[0073] Pupation start time:

[0074] Experimental operation: Count the pupation time of the first larva that successfully pupates in each parallel under each concentration group

[0075] formula:

[0076] End of pupation:

[0077] Experimental operation: Count the pupation time of the last larva that successfully pupates in each parallel under each concentration group

[0078] formula:

[0079] Among the above indicators, all are intuitively result-oriented to characterize the impact of the environment on chironomid larvae. However, in some slightly toxic or complex environments, the chemicals in the environment will not directly harm the chironomid larvae, but will affect and interfere at the tissue and gene levels, which may affect the growth and development of the offspring of the chironomid larvae. Therefore, based on 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.

[0080] In combination with the above content, the following embodiments of the present invention set new evaluation indicators for chironomid larvae, so as to more accurately reflect the development of chironomid larvae.

[0081] Embodiment 1: A method for determining the effect of the environment on the development of chironomid larvae, comprising:

[0082] S101, setting a standard value for the imaginal disc area of ​​chironomid larvae;

[0083] Specifically: obtain a paraffin section of at least one sample of a 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.

[0084] For example, paraffin sections of samples of Chironomid larvae in a normal developmental state are recorded as a control group and subjected to the following treatments (1) to (3).

[0085] (1) Sample collection: Chironomids were cultured continuously for 10 generations in the laboratory. Chironomids were cultured in dechlorinated fresh water with aeration for more than 48 hours, and the pre-culture tank was maintained with mild aeration, temperature of 23±2℃, light-dark ratio of 16:8, and light intensity of 800±200lux. Artificial feed was fed during the breeding period, and 1.0 mg / bird / time was fed three times a week. Before the experiment, the newly laid egg clusters within 8 hours were taken out and cultured separately until the early fourth instar, and samples were taken as larvae for experimental use. The chironomid larvae were placed in a control group (i.e., chironomid larvae in a normal developmental state) in clean water for exposure. When the larvae in the control group developed to the end of the fourth instar, larval samples of the control group and the treatment group were collected on the same day. A 10mL plastic pipette was used when collecting larval samples. The action of sucking the larvae should be gentle and quick to reduce stimulation to the larvae and reduce the mortality of the larvae during sample collection;

[0086] (2) Sample preparation: After collecting the above-mentioned control group (i.e., chironomid larvae in a normal developmental state), select the surviving larvae whose bodies are not damaged during the collection process, and promptly fix the samples in 4% paraformaldehyde. Perform continuous paraffin sections and HE staining on the second to fourth body segments (cross sections) of the larvae. Each sample is cut 10 times in a row (with an interval of 10 μm) to observe the effect of a certain drug on larval development. The purpose of continuous sectioning is to cut the maximum cross-section of the imaginal disc. The more sections there are and the smaller the interval, the more likely it is that the maximum area of ​​the imaginal disc will be observed. Select paraffin sections with complete tissue and clear staining to further measure the maximum area of ​​the cross-section of the imaginal disc of the chironomid larvae (at least five larvae in each group are measured for the imaginal disc).

[0087] (3) Area measurement: A scientific research stereo microscope (Nikon SMZ25) was used to observe and photograph consecutive sections. The position of the imaginal disc in the fourth instar larvae of Chironomids was determined based on all the paraffin sections obtained. The sections with the largest imaginal disc area in each group were selected, and the maximum area of ​​the imaginal disc in each group was measured. The purpose of consecutive paraffin sections and photographing was to select the photos with the largest imaginal disc area. Before observation, the sections were arranged and numbered in the order of the cutting process. Ten consecutive sections were taken for each insect at each concentration. During the sectioning and observation of the imaginal disc of each section, it was necessary to find that the imaginal disc area was in the order of small to large and then to small. The section with the largest imaginal disc area was the maximum area of ​​the imaginal disc of this insect. At this time, a microscope with an area measurement function was used for measurement. Because the imaginal discs were distributed on the left and right sides of the insect body, the two sets of data obtained from measuring the two sides of the insect were calculated during measurement, and no addition was required. It should be noted that the maximum area of ​​the imaginal discs on both sides of the insect body may exist in different sections during the sectioning process. In this case, the maximum area of ​​the corresponding side of the two sections needs to be measured.

[0088] The method for determining the maximum area of ​​the chironomid larvae imaginal disc comprises S1011-S1013:

[0089] S1011, obtaining at least one group of chironomid larvae sample slices containing imaginal discs, wherein the at least one group of chironomid larvae sample slices containing imaginal discs are derived from at least one chironomid larva; the imaginal discs are located on both sides of the center of the chironomid larvae sample slices;

[0090] S1012, selecting a first maximum value from at least one group of first imaginal disc areas located on one side of the center of the chironomid larvae sample slice, and selecting a second maximum value from at least one group of second imaginal disc areas located on the other side of the center of the chironomid larvae sample slice;

[0091] S1013, summing the first maximum value and the second maximum value, and dividing the sum by twice the number of the chironomid larvae to obtain the maximum area of ​​the chironomid larvae imaginal disc.

[0092] S102, calculating the experimental value of the imaginal disc area of ​​at least one chironomid larvae sample in a preset environment, including:

[0093] Obtain a paraffin section of at least one chironomid larvae sample after being affected by a preset environmental environment, determine the maximum area of ​​the chironomid larvae imaginal disc, and then use the maximum area of ​​the chironomid larvae imaginal disc as the experimental value of the chironomid larvae imaginal disc area.

[0094] Exemplarily, bisphenol F is selected to simulate a toxic environment for testing, and the chironomid larvae after the test are taken 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 the following Experimental Examples 1 and 2.

[0095] S103. Based on the comparison between the experimental value of the imago disc area and the standard value of the imago disc area, determine whether the chironomid larvae are affected by the preset environment; when the difference between the standard value of the imago disc area and the standard value of the imago disc area is greater than the standard threshold, determine that the chironomid larvae are promoted by the preset environment and cause overdevelopment; when the difference between the standard value of the imago disc area and the standard value of the imago disc area is less than the standard threshold, determine that the chironomid larvae are inhibited by the preset environment and cause underdevelopment; otherwise, determine that the chironomid larvae are 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.

[0096] Furthermore, when no imaginal disc is observed on one side of the center of the chironomid larvae sample slice and an imaginal disc is observed on the other side of the center of the chironomid larvae sample slice, the area of ​​the imaginal disc on one side of the center of the chironomid larvae sample slice is set to 0 and participates 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 mosquito larvae sample slice, it means that the mosquito larvae sample slice is unusable.

[0097] It should be understood that the principle of the above judgment is: when the insect imaginal disc only exists on one side, and the tissue structure is not found in the other side in the continuous paraffin sections, there are the following possibilities:

[0098] 1. The chironomid larvae were affected by the test substance, and the imaginal disc was malformed and one side was missing. At this time, the imaginal disc area on one side was 0 μm 2 To make calculations, statistics are indispensable;

[0099] 2. Operation error during paraffin sectioning, in which case the section needs to be discarded;

[0100] 3. Because specific segments of the larvae are squeezed or shaken and deformed during experimental operations such as embedding, dehydration, and cutting, the imaginal disc may be displaced to a certain extent, which may result in one or both sides of the imaginal disc being unable to be observed;

[0101] 4. The range of imaginal discs is 2-4 segments of the larvae. This range is only the data obtained by our laboratory through a large number of experiments on early chironomid larvae. There may be slight differences between different species, which needs to be determined according to the organisms and experiments cultured in the laboratory;

[0102] 5. The larvae are too young, so the development of the imaginal disc cannot be observed. In this case, the chironomid larvae selected were at the end of the fourth instar, and the development of the imaginal disc in the control group was better. If the imaginal disc cannot be observed, but it is not due to the above reasons, researchers need to conduct specific analysis based on the specific problem.

[0103] The method for determining the standard threshold value includes:

[0104] The LSD critical value was calculated based on the least significant difference method using the standard value data of the imaginal disc area of ​​chironomid larvae, and the LSD critical value was used as the standard threshold.

[0105] Specifically, the Least Significant Difference (LSD) method is a method used to compare the differences between multiple sample means, especially after analysis of variance (ANOVA), when the ANOVA results show that there are significant differences between at least two groups. The LSD method determines which mean differences between groups are statistically significant by calculating a critical value (i.e., the standard threshold mentioned above).

[0106] Steps to determine the critical value (standard threshold):

[0107] Perform ANOVA analysis: First, you need to perform ANOVA analysis 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.

[0108] Calculate the LSD critical value: After ANOVA confirms the existence of significant differences, the LSD method can be used to further determine which differences between groups are significant; the LSD critical value is usually calculated based on the following formula:

[0109]

[0110] Where: t α / k,N-k is the two-sided α / kQuantile, where N is the total number of observations, k is the number of groups, α is the significance level (such as 0.05); MSE is the Mean Squared Error, obtained from the ANOVA table; and n is the number of samples in each group (assuming that all groups have the same number of samples).

[0111] That is to say, the maximum average area of ​​the imaginal disc of the control group (i.e., the standard value of the imaginal disc area of ​​the chironomid larvae) is used as a reference to compare the maximum average area of ​​the imaginal disc in the treatment group (the experimental value of the imaginal disc area of ​​the chironomid larvae sample), and statistical methods are used to calculate the significance and use specific data to characterize the effect of the test drug on the development of chironomid larvae. Specifically:

[0112] If the maximum area of ​​the insect disc of the treatment group is significantly smaller than the maximum area of ​​the imaginal disc of the control group (i.e., exceeds the standard threshold), it indicates that the development of the larvae in the treatment group is inhibited compared with that in the control group;

[0113] If the maximum area of ​​the insect disc of the treatment group is significantly larger than that of the adult disc of the control group, it means that the development of the larvae in the treatment group is promoted compared with that in the control group;

[0114] If there is no significant difference in the maximum area of ​​the imaginal disc of the treated group compared with the maximum area of ​​the imaginal disc of the control group, and there is no significant difference in appearance and shape, it means that there is no apparent effect on the development of the imaginal disc of the larvae in the treated group compared with the larvae in the control group.

[0115] Experimental Example 1: Prove the feasibility of judging the influence of the environment on the chironomid larvae based on the imaginal disc of the embodiment of the present invention;

[0116] A developmental toxicity experiment of BPF (bisphenol F) on chironomids was conducted. The experimental chironomus tentans was cultured continuously in the laboratory for more than 10 generations. The dechlorinated fresh water was aerated for more than 48 hours for breeding, and the pre-breeding tank was maintained with mild aeration, temperature of 23±2℃, light-dark ratio of 16:8, and light intensity of 800±200lux. During the breeding period, artificial feed (Qingdao LongxingFEED Co, Ltd.) was fed three times a week at 1.0mg / worm / time. The BPF used in this study (purity>99.5%, purchased from Solarbio) was dissolved in DMSO as a mother solution before the experiment, and different doses were added according to the concentration.

[0117] 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 in the acute toxicity experiment, with 4 parallel groups in each group; each parallel group had 5 first-instar larvae. The exposure period was 48 hours, and no feeding or aeration was allowed during the experiment. The inhibition of larvae at 24 hours and 48 hours was recorded, and the acute activity inhibition rate of larvae was calculated. If the larvae did not produce obvious displacement within 15 seconds after gently impacting them with water through a pipette, they were considered to be inhibited;

[0118] Before the chronic toxicity test, newly laid egg clusters within 8 hours were taken out and cultured separately until the early fourth instar, and samples were taken as experimental larvae. Larvae were randomly added to the prepared 50.0 mL BPF experimental solution and the control group without bisphenols (referred to as Control) for exposure. The experimental concentration was set based on the acute toxicity experiment, and the EC 10 ,EC 20 ,EC 50 The larvae were fed with 1.0 mg / day of 1.0 mg / day of 1.5 mg / L of 2.0 mg / L and the aeration was kept gentle during the experiment. The survival and pupation of larvae in each group were counted every 24 hours. The experiment lasted until all larvae pupated or died. Dead larvae were removed every day during the experiment. Additional parallel groups were added to the control group and the 2.0 and 4.0 mg / L treatment groups to collect samples for histopathological analysis of larval imaginal discs.

[0119] like Figure 1 As shown in the table, the acute activity inhibition of Chironomid larvae at 24h and 48h by BPF at various concentrations was 20% after 24h exposure to 4.0mg / L BPF, and increased to 45% after 48h, which significantly inhibited the activity of larvae (P<0.05); when exposed for 24h or more, all tested larvae with concentrations higher than 8.0mg / L were significantly inhibited, with an inhibition rate of more than 95% (P<0.05). The larval inhibition rate was positively correlated with the BPF exposure time and exposure concentration; the effect concentration was positively correlated with the inhibition rate, but negatively correlated with the exposure time (as shown in Table 1).

[0120] Table 1 EC of BPF against Chironomids extensa 10 , E.C. 20 , and EC 20

[0121] Exposure time <![CDATA[EC 10 ]]> <![CDATA[EC 20 ]]> <![CDATA[EC 50 ]]> 24h 1.67mg / L 2.34mg / L 4.17mg / L 48h 1.31mg / L 1.87mg / L 3.44mg / L

[0122] like Figure 2As shown in Figure 2, during the exposure period, all larvae in the control group survived and developed normally to the pupation stage. The mortality rate of larvae 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 between the larvae in the control group and the control group (P>0.05). When the BPF concentration was ≥2.0 mg / L, the larvae mortality rate was significantly higher than that in the control group (P<0.05). Figure 2 (shown in A).

[0123] The distribution of larval pupation time and cumulative pupation rate under BPF exposure are shown in Figure 2B. Compared with the control group, the presence of BPF delayed larval pupation; the cumulative pupation rate decreased with increasing concentration. When the BPF concentration was ≤1.5mg / L, the cumulative pupation rate showed a decreasing trend, although it was not significant (P>0.05). When the BPF concentration was ≥2.0mg / L, the cumulative pupation rate of larvae in all treatment groups was significantly reduced (P<0.05) (e.g. Figure 2 (shown in B).

[0124] Effects of BPF on the time of larval pupation and the duration of each stage Figure 2 As shown in C and E. Compared with the control group, 3.0 mg / L BPF significantly delayed the average start time of pupation (P<0.05), and there was a delay trend in other exposure groups (P>0.05); however, the presence of BPF did not lead to statistically significant changes in the average pupation time and the average end time of pupation, although these parameters fluctuated at different concentrations (P>0.05);

[0125] To explore the potential effects of BPF exposure on larval development, the location of the imaginal discs (green marks) in the fourth instar larvae of Chironomids expansus was determined by referring to the identification of some organ tissues of Aedes aegypti. Figure 3 E, J, and O correspond to the maximum area of ​​the imaginal disc of the fourth instar larvae in the control group, 2.0 mg / L, and 4.0 mg / L treatment groups, respectively. The development of the imaginal disc was quantified by measuring the area of ​​the imaginal disc in serial sections. Figure 3 As shown in B. Compared with the control group, the imaginal disc area of ​​the 4.0 mg / L BPF treatment group was significantly smaller than that of the control group (P<0.05), and the inhibition of 4.0 mg / L BPF on the development of larval imaginal disc was more obvious than that of the 2.0 mg / L group.

[0126] In summary, the embodiment of the present invention uses the maximum area of ​​the imaginal disc of chironomid larvae as one of the indicators to confirm the effect of BPF on the growth and development of chironomid larvae. The results show that the method is time-saving, simple to operate, and convenient and fast to process samples. It is confirmed that 4.0 mg / L BPF can cause significant inhibition on the development of the imaginal disc of chironomid larvae.

[0127] Furthermore, the above indicators may not be able to accurately characterize the tissues and development of some chironomid larvae, such as the following test:

[0128] Experimental Example 2: Prove the superiority of the embodiment of the present invention in judging the influence of the environment on the chironomid larvae based on the imaginal disc;

[0129] Reproductive toxicity: A total of three exposure concentrations were set, and the highest concentration was set based on the minimum effect concentration in the developmental toxicity test, namely, 0.5, 1.0, and 2.0 mg / L treatment groups and 0 mg / L control group, with 3 parallel groups in each group and 20 fourth-instar larvae in each parallel. The experimental environmental conditions were consistent with those in 2.2(2). During the exposure period, the emergence of Chironomid larvae in the control group and the three treatment groups was continuously observed. The newly emerged adults of each experimental group were collected twice a day at 08:00 and 20:00 and transferred to a new 5L glass beaker filled with aerated fresh water according to the male and female ratios. Healthy male and female adults were moved in at a ratio of 1:1 to determine the reproductive capacity of infected female and male insects, and the egg-laying rate of each group was recorded.

[0130] The egg-laying rate was calculated by the number of egg clusters laid by each female mosquito. The egg-laying rate was the total number of egg clusters laid in each experimental container divided by the total number of female mosquitoes that survived and did not suffer any damage in the experimental container. Additional parallel groups were added to the control and treatment groups to simultaneously measure the expression of genes related to the ecdysone, juvenile hormone, and insulin pathways in adults that had just emerged for less than 12 hours.

[0131] Experimental results:

[0132] like Figure 4 As shown, the effect of BPF on the egg-laying rate of chironomids (individual level - no effect on the egg-laying rate of female adults)

[0133] The effect of reproductive toxicity BPF on the egg-laying rate of chironomids treated with different concentrations is shown in Fig. 5A-B. Compared with the control group, the egg-laying rate of the infected female chironomids × the uninfected male chironomids in the four BPF treatment groups was 85%-95%, with no significant difference (P>0.05). The egg-laying rate of infected male chironomids × uninfected female chironomids was 93% in the 0.5 mg / L treatment group, which was not significantly different from that in the control group (P>0.05); the egg-laying rates of the 1.0 mg / L and 2.0 mg / L treatment groups were 75% and 61%, respectively, which were significantly lower than those in the control group (P<0.01).

[0134] In summary, the above-mentioned reproduction experiment characterized the effect of BPF on female chironomid adults. The experiment showed that no significant difference was found in the reproduction indicators, that is, BPF had no significant effect on the reproduction of chironomids, but for the imaginal disc area in the embodiment of the present invention, there was a significant difference compared with the control group, indicating that some genes were significantly upregulated at the genetic level; it was proved that the use of imaginal 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 determining the effect of the environment on the development of chironomid larvae, characterized in that: include: Set the standard value of the imaginal disc area of ​​chironomid larvae; Calculate the experimental value of the imaginal disc area of ​​at least one chironomid larvae sample in a preset environment; Based on the comparison between the experimental value of the imaginal disc area and the standard value of the imaginal disc area, it is judged whether the chironomid larvae are affected by the preset environment; when the difference between the standard value of the imaginal disc area and the standard value of the imaginal disc area is greater than the standard threshold value, it is judged that the chironomid larvae are promoted by the preset environment and cause overdevelopment; when the difference between the standard value of the imaginal disc area and the standard value of the imaginal disc area is less than the standard threshold value, it is judged that the chironomid larvae are inhibited by the preset environment and cause underdevelopment; otherwise, it is judged that the chironomid larvae are not affected by the preset environment.

2. A method for determining the effect of the environment on the development of chironomid larvae according to claim 1, characterized in that: The method for setting the standard value of the imaginal disc area of ​​chironomid larvae comprises: Obtain a paraffin section of at least one sample of a 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.

3. A method for determining the effect of the environment on the development of chironomid larvae according to claim 2, characterized in that: The method for calculating the experimental value of the imaginal disc area of ​​at least one chironomid larvae sample in a preset environment comprises: Obtain a paraffin section of at least one chironomid larvae sample after being affected by a preset environmental environment, determine the maximum area of ​​the chironomid larvae imaginal disc, and then use the maximum area of ​​the chironomid larvae imaginal disc as the experimental value of the chironomid larvae imaginal disc area.

4. A method for determining the effect of the environment on the development of chironomid larvae according to any one of claims 2 or 3, characterized in that: The method for determining the maximum area of ​​the imaginal disc of the chironomid larvae comprises: Obtain at least one group of chironomid larvae sample slices containing imaginal discs, wherein the at least one group of chironomid larvae sample slices containing imaginal discs are derived from at least one chironomid larva; the imaginal discs are located on both sides of the center of the chironomid larvae sample slices; A first maximum value is selected from at least one group of first imaginal disc areas located on one side of the center of the chironomid larvae sample slice, and a second maximum value is selected from at least one group of second imaginal disc areas located on the other side of the center of the chironomid larvae sample slice; The first maximum value and the second maximum value are summed and then divided by twice the number of the chironomid larvae to obtain the maximum area of ​​the chironomid larvae imaginal disk.

5. A method for determining the effect of the environment on the development of chironomid larvae according to claim 4, characterized in that: The method for determining the standard threshold value includes: The LSD critical value was calculated based on the least significant difference method using the standard value data of the imaginal disc area of ​​chironomid larvae, and the LSD critical value was used as the standard threshold.

6. A method for determining the effect of the environment on the development of chironomid larvae according to claim 5, characterized in that: When no imaginal disk is observed on one side of the center of the chironomid larvae sample slice and an imaginal disk is observed on the other side of the center of the chironomid larvae sample slice, the area of ​​the imaginal disk on one side of the center of the chironomid larvae sample slice is set to 0 and is involved in the calculation of the experimental value of the imaginal disk area.

7. A method for determining the effect of the environment on the development of chironomid larvae according to claim 1, characterized in that: The preset environment includes a water environment or a water sediment environment, and the water environment or the water sediment environment contains bisphenol compounds.

8. A method for determining the effect of the environment on the development of chironomid larvae according to claim 1, characterized in that: include: S101, setting a standard value for the imaginal disc area of ​​chironomid larvae; Obtain a paraffin section of at least one sample of a 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; The method of obtaining a paraffin section of at least one chironomid larvae sample in a normal developmental state and determining the maximum area of ​​the imaginal disc of the chironomid larvae comprises (1) to (3); (1) Sample collection: Take a plurality of chironomids, culture the chironomids in the laboratory for 10 generations, and then culture the chironomids after 10 generations in aerated dechlorinated fresh water for 48 hours, 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 breeding period, feed three times a week at 1.0 mg / bird / time, until a plurality of chironomid egg strings newly produced within 8 hours are obtained, and the chironomid egg strings are cultured separately to the early stage of the fourth instar and sampled 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 stage of the fourth instar, use a 10 mL plastic pipette to collect them in the clean water to obtain a plurality of chironomid larvae samples; (2) Sample preparation: Among the plurality of chironomid larvae samples, the chironomid larvae that are alive and intact are fixed in 4% paraformaldehyde to obtain a plurality of fixed chironomid larvae, and the second to fourth body segments of the fixed chironomid larvae are continuously cross-sectioned 10 times at intervals of 10 μm and stained with HE to obtain a chironomid larvae sample section with the largest cross section of the imaginal disc; (3) Area measurement: using a scientific research-grade stereo microscope to observe the chironomid larvae sample slices, determine the position of the imaginal disc in the fourth-instar chironomid larvae, select the chironomid larvae sample slices with the largest area of ​​the chironomid larvae imaginal disc, and determine the maximum area of ​​the chironomid larvae imaginal disc; Wherein, the method for determining the maximum area of ​​the imaginal disc of the chironomid larvae comprises S1011-S1013: S1011, obtaining at least one group of chironomid larvae sample slices containing imaginal discs, wherein the at least one group of chironomid larvae sample slices containing imaginal discs are derived from at least one chironomid larva; the imaginal discs are located on both sides of the center of the chironomid larvae sample slices; S1012, selecting a first maximum value from at least one group of first imaginal disc areas located on one side of the center of the chironomid larvae sample slice, and selecting a second maximum value from at least one group of second imaginal disc areas located on the other side of the center of the chironomid larvae sample slice; S1013, summing the first maximum value and the second maximum value, and dividing the sum by twice the number of the chironomid larvae to obtain the maximum area of ​​the chironomid larvae imaginal disc; S102, calculating the experimental value of the imaginal disc area of ​​at least one chironomid larvae sample in a preset environment, including: Obtaining a paraffin section of at least one chironomid larvae sample after being affected by a preset environmental environment, determining the maximum area of ​​the chironomid larvae imaginal disc, and then using the maximum area of ​​the chironomid larvae imaginal disc as an experimental value of the chironomid larvae imaginal disc area; S103, based on the comparison between the experimental value of the imago disc area and the standard value of the imago disc area, judging whether the chironomid larvae are affected by the preset environment; when the difference between the standard value of the imago disc area and the standard value of the imago disc area is greater than the standard threshold, judging that the chironomid larvae are promoted by the preset environment and cause overdevelopment; when the difference between the standard value of the imago disc area and the standard value of the imago disc area is less than the standard threshold, judging that the chironomid larvae are inhibited by the preset environment and cause underdevelopment; otherwise, judging that the chironomid larvae are not affected by the preset environment; The method for determining the standard threshold value includes: The standard threshold is calculated based on the least significant difference method using the standard value data of the imaginal disc area of ​​the chironomid larvae; The standard threshold is calculated according to the following formula: Where: t α / k,N-k is the two-sided α / k quantile of the t-distribution with Nk degrees of freedom, where N is the total number of observations of paraffin sections of chironomid larvae, k is the number of chironomid larvae, each of which contains a set of chironomid larvae sample sections, α is the significance level; MSE is the mean square error; and n is the number of paraffin sections in each chironomid larva.

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