CN110672648A - Method for distinguishing different resource groups of non-lethal coilia ectenes - Google Patents
Method for distinguishing different resource groups of non-lethal coilia ectenes Download PDFInfo
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- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
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Abstract
The invention relates to a method for distinguishing different resource groups of non-lethal coilia ectenes, which comprises the following steps: collecting, cleaning, embedding, cutting, polishing, cleaning, coating and analyzing a sample of the chest fin of the coilia ectenes. The invention takes the pectoral fin strip as a research object, and overcomes the defects of lethality and destructiveness on the basis of meeting the high accuracy and objectivity consistent with the otolith microchemical analysis technology. The tested individuals can objectively, accurately and obviously distinguish whether the individuals belong to a migration resource group. In addition, the invention combines the characteristics of fish hard tissues and innovatively selects and uses the chest fin ray, so that the difficulty and time for preparing samples in the early stage and the conditions and time for analyzing samples in the later stage are greatly improved.
Description
Technical Field
The invention relates to a method for distinguishing different resource groups of non-lethal coilia ectenes, belonging to the technical field of methods for distinguishing different resource groups of aquatic wild animals.
Background
Coilia ectenes (Coilia nasus) As the name of ChinaOne representative noble fish has resources facing the current situation of lofty risk, and the related protection is not slow. No. 5 announced that the release of special fishing licenses such as coilia ectenes (Yangtze river coilia ectenes) was determined to be stopped in 2018 of the rural agricultural department, and the productive fishing of natural resources of coilia ectenes is prohibited. Therefore, the strength of the country for protecting the coilia ectenes resources can be seen. It is worth noting that the ecotype composition of coilia ectenes is very complicated in our country. Coilia ectenes with both retroversion type and freshwater type (C. brachygnathus) And lake anchovy (C. nasus taihuensis). The first kind of population has the highest value and the lowest resource amount and needs to be effectively protected, and the other two kinds of population are widely distributed in lakes along the Yangtze river or even in the river section, so that part of water areas even become dominant populations. Due to the similar appearance and the frequent occurrence of mixed habitation among different resource groups, especially some recent research reports further overturn the traditional experience of using the traditional appearance to identify migration or not. This makes it difficult to evaluate the current situation of resources of the migration type coilia ectenes and to accurately locate the important habitat of the migration type population ("three fields and one channel"). The method brings difficulty for reasonable evaluation and effective protection of coilia ectenes resource groups.
With the development of interdiscipline, the earlap microchemical "fingerprint" technology has been proved by more and more researches as an effective method for reconstructing the migration habit, habitat history and group association of the anchovy fishes. Based on the technology, the problem of the coilia ectenes migration ecology can be solved one by one. Especially, the important advantages in inverting the history of fish habitat are becoming one of the important techniques for identifying whether the fish is a migration type fish. However, it should be noted that when otoliths are located in the fish inner ear labyrinth, the sampling process necessarily results in death of the fish body and destruction of the head tissue. Therefore, the search for a material which can be obtained quickly (even non-lethal) and can be effectively used as an otolith substitute material for related research is undoubtedly of great significance.
Due to the situations, a tool for conveniently and accurately distinguishing the species of the anchovy fishes is urgently needed to be found. Based on the above consideration, the present invention utilizes pectoral fin strips, which are also hard tissues, as analysis objects and performs element content analysis. The method is efficient, accurate, rapid and non-lethal for identifying different coilia nasus resource groups, so that technical support is provided for accurately evaluating coilia nasus resource conditions and developing effective protection work.
Disclosure of Invention
The invention aims to overcome the defects of lack of accuracy and high lethality and destructiveness of other methods in the traditional method, and provides a non-lethal method for discriminating different resource groups of coilia ectenes, which can effectively and accurately discriminate different resource groups of coilia ectenes under the conditions of non-lethality and low damage.
According to the technical scheme provided by the invention, the method for distinguishing different resource groups of the non-lethal coilia ectenes comprises the following steps:
step (1): picking up the chest fin strips of the coilia ectenes as a research object;
step (2): firstly, removing organic tissues on the surface of the pectoral fin strip of the coilia ectenes by using absolute alcohol, then rinsing the coilia ectenes pectoral fin strip by using purified water, and finally putting the coilia ectenes pectoral fin strip into an oven for drying;
uniformly mixing epoxy resin and triethylene tetramine in a weight ratio of 6 ~ 7:1 for 5 ~ 10 minutes to form embedding resin, integrally embedding the coilia ectenes pectoral fin strips by using the embedding resin to prevent air bubbles from being generated in the embedding resin and between the embedding resin and the coilia ectenes pectoral fin strips, and finally putting the coilia ectenes pectoral fin strips integrally embedded by the embedding resin into a drying oven for drying, and obtaining a sample after the drying is finished;
taking out the sample, cutting off redundant resin at the bottom until one end of the pectoral fin strip is exposed, vertically sticking the whole sample on the glass slide with glue with the exposed surface of the pectoral fin strip downwards, cutting off redundant sample after the glue is solidified, and only keeping the pectoral sample with the height of 3 ~ 5 mm fixed on the glass slide;
and (5): firstly, grinding the upper end face of a sample by using a grinding machine, then polishing the upper end face of the sample by using silicon carbide polishing solution and flannelette, and confirming that the wheel pattern is clear under a metallographic microscope and the surface has no obvious scratch to obtain a sample slice;
ultrasonically cleaning the sample slice by using purified water, and airing the sample slice at room temperature after ultrasonic cleaning, wherein the frequency of ultrasonic waves is 15 ~ 30 kilohertz, the power of the ultrasonic waves is 300 ~ 500 watts, and the ultrasonic cleaning time is controlled to be 3 ~ 5 minutes;
and (7): carrying out carbon film evaporation on the sample slice;
and (8): and (3) carrying out electronic probe quantitative analysis and surface distribution analysis on the sample slice, confirming the distribution characteristics of strontium elements and calcium elements on the section of the sample slice, and judging the resource group to which the sample slice belongs according to the distribution characteristics.
Preferably, the method comprises the following steps: in the step (1), the first pectoral fin strip on the left side of the coilia ectenes is taken as a research object.
Preferably, in the step (2), the oven temperature is controlled at 30 ~ 35 ℃ and the drying time is controlled at 1 ~ 2 hours.
Preferably, in the step (3), the oven temperature is controlled at 30 ~ 36 ℃ and the drying time is controlled at 6 ~ 10 hours.
Preferably, in step (5), the sample sheet has a thickness of 300 ~ 500 microns.
Preferably, in the step (7), the current of the vapor deposition coating film is controlled to be 20 ~ 30A, and the time of the vapor deposition coating film is controlled to be 20 ~ 40 s.
Preferably, in the step (8), the conditions for the quantitative analysis are an acceleration voltage of 15 ~ 20kV and an electron beam current of 1.5 ~ 2X 10-8A. The diameter of the beam spot is 2 ~ 3 μm.
Preferably, in the step (8), the surface distribution analysis is performed under the conditions of an acceleration voltage of 15 ~ 20kV and an electron beam current of 3 ~ 4X 10-7A, the diameter of the beam spot is 2 ~ 3 μm.
Preferably, the method comprises the following steps: in the step (8), the quantitative analysis result of the freshwater coilia ectenes is uniform, and the surface distribution analysis result shows stable blue; the migration type coilia ectenes quantitative analysis result shows a plurality of obvious stages, the surface distribution analysis result core area is blue, and then a green or even yellow annular strip representing high strontium concentration exists.
The invention takes the pectoral fin strip as a research object, and overcomes the defects of lethality and destructiveness on the basis of meeting the high accuracy and objectivity consistent with the otolith microchemical analysis technology. The tested individuals can objectively, accurately and obviously distinguish whether the individuals belong to a migration resource group. In addition, the invention combines the characteristics of fish hard tissues and innovatively selects and uses the chest fin ray, so that the difficulty and time for preparing samples in the early stage and the conditions and time for analyzing samples in the later stage are greatly improved.
Drawings
FIG. 1 is a diagram of a sample wafer after polishing in accordance with the present invention.
FIG. 2 shows the result of quantitative analysis of the pectoral fin strips of the coilia ectenes of the Yangtze river mouth in example 1.
Fig. 3 shows the analysis result of the pectoral fin surface distribution of the coilia ectenes of the Yangtze river mouth in example 1.
FIG. 4 shows the result of quantitative analysis of the pectoral fin strips of coilia ectenes from example 2.
FIG. 5 shows the analysis results of the pectoral fin surface distribution of the coilia ectenes in example 2.
FIG. 6 shows the quantitative analysis results of the chest fin strip of the Yanghu coilia ectenes of example 3 .
FIG. 7 shows the analysis results of the fin surface distribution of the Yanghu coilia ectenes of example 3 .
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1
An coilia fish population distinguishing method based on the form of otolith adopts the following process steps:
collecting in step (1): collecting individual coilia ectenes at the mouth of a Yangtze river in 2018, and picking a first pectoral fin strip on the left side of the individual coilia ectenes as a research object;
cleaning in step (2): removing organic tissues such as muscles on the surface of the chest fin ray by using absolute ethyl alcohol, rinsing by using purified water, and then putting into an oven for drying, wherein the temperature of the oven is controlled at 30 ℃, and the drying time is controlled at 2 hours;
embedding in step (3): uniformly mixing epoxy resin and triethylene tetramine for 5 minutes in a weight ratio of 6:1, firstly, dropwise adding a small amount of epoxy resin and triethylene tetramine to the periphery of a chest fin, picking out and removing bubbles by using a toothpick, continuously adding the epoxy resin and the triethylene tetramine to cover a chest fin sample, finally, putting the chest fin sample into a 30 ℃ oven for solidification, controlling the drying time to be 10 hours, and obtaining the sample after the drying is finished;
cutting, namely taking out a sample, cutting off redundant resin blocks at the bottom until the pectoral fin strip sample is exposed, vertically sticking the whole resin blocks on a glass slide by using AB glue with the exposed surface of the pectoral fin strip downwards, cutting off the redundant resin blocks after solidification, and only reserving a 3 ~ 5 mm sample fixed on the glass slide;
grinding, namely grinding a sample by using a grinding machine (the particle size is 20 ~ 50 mu m), then polishing by using silicon carbide polishing solution and flannelette, and determining that the wheel pattern is clear under a metallographic microscope and no obvious scratch is formed on the surface of the wheel pattern, thus obtaining a sample slice, wherein the thickness of the sample slice is 300 ~ 500 microns;
and (6) cleaning: ultrasonically cleaning a sample slice by using purified water, and airing the sample slice at room temperature after ultrasonic cleaning; the frequency of the ultrasonic wave is 15 kilohertz, the power of the ultrasonic wave is 300 watts, and the ultrasonic cleaning time is controlled to be 3 minutes;
step (7) film coating: before sample analysis, a carbon film is required to be evaporated, the current of an evaporated coating is controlled at 20A, and the time of the evaporated coating is controlled at 40 seconds;
step (8) analysis: and (3) carrying out electronic probe quantitative analysis and surface distribution analysis on the coated sample, and confirming the element distribution characteristics on the section of the sample, wherein the conditions of the quantitative analysis are as follows: accelerating voltage of 15kV and electron beam current of 1.5 × 10-8A. The diameter of the beam spot is 2 μm; the conditions for the area distribution analysis were: acceleration voltage 15kV, electron beam current 3X 10-7A, the diameter of a beam spot is 2 micrometers, the quantitative line analysis result shows that three obvious stages are provided, namely 0 ~ 44 micrometers, the Sr/Ca ratio is 2.01 +/-0.27, 44 ~ 74 micrometers and 3.71 +/-0.32, and the edge is 2.27 (Mann-Whitney U-test), the surface distribution analysis result shows that the blue color with low Sr content in the core area, the green ring zone with high Sr concentration at the periphery and the blue ring zone at the edge part are typical of the swimming type coilia ectenes.
Example 2
An coilia fish population distinguishing method based on the form of otolith adopts the following process steps:
collecting in step (1): collecting individual coilia ectenes in the Taihu lake in 2018, and picking a first pectoral fin strip on the left side of the individual coilia ectenes as a research object;
cleaning in step (2): removing organic tissues such as muscles on the surface of the chest fin ray by using absolute ethyl alcohol, rinsing by using purified water, and then putting into an oven for drying, wherein the temperature of the oven is controlled at 35 ℃, and the drying time is controlled at 1 hour;
embedding in step (3): uniformly mixing epoxy resin and triethylene tetramine in a weight ratio of 7:1 for 10 minutes, firstly, dropwise adding a small amount of epoxy resin and triethylene tetramine to the periphery of a chest fin, picking out and removing bubbles by using a toothpick, continuously adding the epoxy resin and the triethylene tetramine to cover a chest fin sample, finally, putting the chest fin sample into a 36 ℃ oven for solidification, controlling the drying time to be 6 hours, and obtaining the sample after the drying is finished;
cutting, namely taking out the solidified resin blocks, cutting off redundant resin blocks at the bottoms until pectoral fin strip samples are exposed, vertically sticking the whole resin blocks on a glass slide with AB glue downwards by using the exposed surfaces of the pectoral fin strips, cutting off the redundant resin blocks after solidification, and only reserving the 3 ~ 5 mm samples fixed on the glass slide;
grinding, namely grinding a sample by using a grinding machine (the particle size is 20 ~ 50 mu m), then polishing by using silicon carbide polishing solution and flannelette, and determining that the wheel pattern is clear under a metallographic microscope and no obvious scratch is formed on the surface of the wheel pattern, thus obtaining a sample slice, wherein the thickness of the sample slice is 300 ~ 500 microns;
and (6) cleaning: ultrasonically cleaning a sample slice by using purified water, and airing the sample slice at room temperature after ultrasonic cleaning; the frequency of the ultrasonic wave is 30 kilohertz, the power of the ultrasonic wave is 500 watts, and the ultrasonic cleaning time is controlled to be 5 minutes;
step (7) film coating: before sample analysis, a carbon film is required to be evaporated, the current of an evaporated coating is controlled at 30A, and the time of the evaporated coating is controlled at 20 seconds;
step (8) analysis: and (3) carrying out electronic probe quantitative analysis and surface distribution analysis on the coated sample, and confirming the element distribution characteristics on the section of the sample, wherein the conditions of the quantitative analysis are as follows: acceleration voltage of 20kV and electron beam current of 2 x 10-8A. The diameter of the beam spot is 3 μm; the conditions for the area distribution analysis were: acceleration voltage 20kV and electron beam current 4X 10-7A, selecting a beam spot with the diameter of 3 μm; the results of the quantitative line analysis showed that the fluctuations stabilized at 2.04. + -. 0.74. The surface distribution analysis result shows that the Sr content is uniform blue, and the coilia ectenes is typical of the freshwater type coilia ectenes.
Example 3
An coilia fish population distinguishing method based on the form of otolith adopts the following process steps:
collecting in step (1): collecting individual coilia ectenes in the Poyang lake in 2018, and picking the first pectoral fin strip on the left side of the individual coilia ectenes as a research object;
cleaning in step (2): removing organic tissues such as muscles on the surface of the chest fin ray by using absolute ethyl alcohol, rinsing by using purified water, and then putting into an oven for drying, wherein the temperature of the oven is controlled at 33 ℃, and the drying time is controlled at 1.5 hours;
embedding in step (3): uniformly mixing epoxy resin and triethylene tetramine in a weight ratio of 6.5:1 for 8 minutes, firstly, dropwise adding a small amount of epoxy resin and triethylene tetramine to the periphery of a chest fin strip, picking out and removing bubbles by using a toothpick, continuously adding the mixture until the mixture covers the chest fin strip sample, finally, putting the mixture into a 33 ℃ oven for solidification, controlling the drying time to be 8 hours, and obtaining the sample after the drying is finished;
cutting, namely taking out the solidified resin blocks, cutting off redundant resin blocks at the bottoms until pectoral fin strip samples are exposed, vertically sticking the whole resin blocks on a glass slide with AB glue downwards by using the exposed surfaces of the pectoral fin strips, cutting off the redundant resin blocks after solidification, and only reserving the 3 ~ 5 mm samples fixed on the glass slide;
grinding, namely grinding a sample by using a grinding machine (the particle size is 20 ~ 50 mu m), then polishing by using silicon carbide polishing solution and flannelette, and determining that the wheel pattern is clear under a metallographic microscope and no obvious scratch is formed on the surface of the wheel pattern, thus obtaining a sample slice, wherein the thickness of the sample slice is 300 ~ 500 microns;
and (6) cleaning: ultrasonically cleaning a sample slice by using purified water, and airing the sample slice at room temperature after ultrasonic cleaning; the frequency of the ultrasonic wave is 20 kilohertz, the power of the ultrasonic wave is 400 watts, and the ultrasonic cleaning time is controlled to be 4 minutes;
step (7) film coating: before sample analysis, a carbon film is required to be evaporated, the current of an evaporated coating is controlled at 25 amperes, and the time of the evaporated coating is controlled at 30 seconds;
step (8) analysis: carrying out electronic probe quantitative analysis and surface distribution analysis on the coated sample, and confirming element distribution characteristics on the section of the sample; wherein, the quantitative analysis conditions are as follows: acceleration voltage of 18kV and electron beam current of 1.7 × 10-8A. The diameter of the beam spot is 2.5 μm; the conditions for the area distribution analysis were: acceleration voltage of 17kV and electron beam current of 3.5 × 10-7A, the diameter of a beam spot is 2.5 μm; the results of the quantitative line analysis showed that the fluctuations stabilized at 1.37. + -. 0.51. The surface distribution analysis result shows that the Sr content is uniform blue, and the coilia ectenes is typical of the freshwater type coilia ectenes.
Claims (9)
1. A method for discriminating different resource groups of non-lethal coilia ectenes is characterized by comprising the following steps:
step (1): picking up the chest fin strips of the coilia ectenes as a research object;
step (2): firstly, removing organic tissues on the surface of the pectoral fin strip of the coilia ectenes by using absolute alcohol, then rinsing the coilia ectenes pectoral fin strip by using purified water, and finally putting the coilia ectenes pectoral fin strip into an oven for drying;
uniformly mixing epoxy resin and triethylene tetramine in a weight ratio of 6 ~ 7:1 for 5 ~ 10 minutes to form embedding resin, integrally embedding the coilia ectenes pectoral fin strips by using the embedding resin to prevent air bubbles from being generated in the embedding resin and between the embedding resin and the coilia ectenes pectoral fin strips, and finally putting the coilia ectenes pectoral fin strips integrally embedded by the embedding resin into a drying oven for drying, and obtaining a sample after the drying is finished;
taking out the sample, cutting off redundant resin at the bottom until one end of the pectoral fin strip is exposed, vertically sticking the whole sample on the glass slide with glue with the exposed surface of the pectoral fin strip downwards, cutting off redundant sample after the glue is solidified, and only keeping the pectoral sample with the height of 3 ~ 5 mm fixed on the glass slide;
and (5): firstly, grinding the upper end face of a sample by using a grinding machine, then polishing the upper end face of the sample by using silicon carbide polishing solution and flannelette, and confirming that the wheel pattern is clear under a metallographic microscope and the surface has no obvious scratch to obtain a sample slice;
ultrasonically cleaning the sample slice by using purified water, and airing the sample slice at room temperature after ultrasonic cleaning, wherein the frequency of ultrasonic waves is 15 ~ 30 kilohertz, the power of the ultrasonic waves is 300 ~ 500 watts, and the ultrasonic cleaning time is controlled to be 3 ~ 5 minutes;
and (7): carrying out carbon film evaporation on the sample slice;
and (8): and (3) carrying out electronic probe quantitative analysis and surface distribution analysis on the sample slice, confirming the distribution characteristics of strontium elements and calcium elements on the section of the sample slice, and judging the resource group to which the sample slice belongs according to the distribution characteristics.
2. The method for discriminating between different resource groups of non-lethal coilia ectenes according to claim 1, wherein: in the step (1), the first pectoral fin strip on the left side of the coilia ectenes is taken as a research object.
3. The method for discriminating different resource groups of non-lethal coilia ectenes according to claim 1, wherein in the step (2), the oven temperature is controlled to be 30 ~ 35 ℃ and the drying time is controlled to be 1 ~ 2 hours.
4. The method for discriminating different resource groups of non-lethal coilia ectenes according to claim 1, wherein in the step (3), the oven temperature is controlled to be 30 ~ 36 ℃ and the drying time is controlled to be 6 ~ 10 hours.
5. The method for discriminating different resource groups of non-lethal coilia ectenes according to claim 1, wherein the thickness of the sample sheet in the step (5) is 300 ~ 500 μm.
6. The method for discriminating different resource groups of non-lethal coilia ectenes according to claim 1, wherein in the step (7), the current of the vapor deposition coating is controlled to be 20 ~ 30A, and the time of the vapor deposition coating is controlled to be 20 ~ 40 s.
7. The non-lethal coilia ectenes diverse resource pool of claim 1The body discrimination method is characterized in that in the step (8), the quantitative analysis conditions are that the acceleration voltage is 15 ~ 20kV, and the electron beam current is 1.5 ~ 2X 10-8A. The diameter of the beam spot is 2 ~ 3 μm.
8. The method for discriminating different resource groups of non-lethal coilia ectenes according to claim 1, wherein the conditions of the surface distribution analysis in the step (8) are an acceleration voltage of 15 ~ 20kV and an electron beam current of 3 ~ 4X 10-7A, the diameter of the beam spot is 2 ~ 3 μm.
9. The method for discriminating between different resource groups of non-lethal coilia ectenes according to claim 1, wherein: in the step (8), the quantitative analysis result of the freshwater coilia ectenes is uniform, and the surface distribution analysis result shows stable blue; the migration type coilia ectenes quantitative analysis result shows a plurality of obvious stages, the surface distribution analysis result core area is blue, and then a green or even yellow annular strip representing high strontium concentration exists.
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| CN113552153A (en) * | 2021-07-27 | 2021-10-26 | 中国水产科学研究院淡水渔业研究中心 | Strontium finlet marking method for non-lethal freshwater fish and detection method thereof |
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