Accurate measuring method for hardness of crassostrea gigas shell and application

Abstract

The invention discloses a standardized evaluation method for shell hardness of crassostrea gigas, and belongs to the field of aquatic shellfish mechanical phenotype determination and biomineralization material performance detection. The method comprises the following steps: carrying out cleaning pretreatment on double shells of crassostrea gigas; respectively determining the longest axis and the widest axis in the contours of the left shell and the right shell, constructing an orthogonal reference cross axis, axially arranging three standardized measurement sites of the left shell and the right shell at positions about 5mm away from the inner sides of the edges of the shells, and marking the standardized measurement sites; cutting the marked area to obtain a flat shell sample; applying a load of about 10 gf to each measurement site by using a Vickers microhardness tester, maintaining the pressure for about 10 seconds, and carrying out multiple indentation tests to obtain an original hardness value; carrying out abnormal value elimination and mean value calculation on the data to obtain representative hardness values of all sites; and further comprehensively calculating representative hardness values of six sites of the left shell and the right shell, and constructing an individual comprehensive shell hardness index. Compared with an existing single-point or random sampling measurement method, the method has the advantages that the problems of irregular shell shapes, asymmetry of left shells and right shells and the like of oysters are considered, and the accuracy and repeatability of oyster hardness phenotype measurement are remarkably improved through standardization of measurement sites and multi-site high-repetition measurement. The method can be widely applied to the fields of oyster stress resistance evaluation, marine acidification, environmental monitoring and the like, provides possibility for accurate phenotype determination in GWAS and QTL positioning in breeding, and has a huge application prospect.

Description

Technical Field

[0001] This invention relates to the field of shellfish mechanical property testing and evaluation technology, specifically to a standardized method for measuring and comprehensively evaluating the hardness of oyster shells, belonging to the fields of mechanical testing of aquaculture biomaterials, analysis of shell micromechanical properties, and environmental adaptability assessment. Background Technology

[0002] Shell hardness, as an indicator of the mechanical properties of shellfish, is increasingly recognized as an important indicator for assessing the resilience of shellfish to acidification and the stress of marine environments, especially with the intensification of global ocean acidification and ecological changes. This is particularly true for Pacific oysters (Crassostrea gigas). Crassostrea gigas For aquaculture species with high economic value, such as , hard shells can effectively resist physical wear and tear in the aquaculture environment, predation attacks by predators, and mechanical damage during harvesting, processing, and transportation, thereby significantly improving the survival rate and commodity integrity. Increasing shell hardness can generate huge economic value.

[0003] As a typical biomineralized composite material, oyster shells have a complex structure, composed of calcium carbonate crystals, an organic matrix, and multi-layered microstructures. At the microscale, oyster shell hardness exhibits significant spatial heterogeneity, with marked differences in mineralization degree, crystal arrangement, microlayer structure, and organic matrix content across different regions. This natural structural heterogeneity leads to substantial variations in mechanical properties across different areas of the shell, making hardness measurements susceptible to the influence of the measurement site selection.

[0004] In existing technologies, traditional shell hardness testing methods mostly employ single-point testing or random sampling. Due to the lack of unified site standards and systematic sampling strategies, the measurement results are often significantly affected by local microstructural variations, resulting in large data fluctuations and insufficient repeatability and stability. For oysters, which have asymmetrical shells and high heterogeneity, single-point measurements cannot accurately reflect the overall mechanical performance level of the individual. Therefore, establishing a method that, under feasible conditions, can systematically overcome the influence of shell micromechanical heterogeneity, achieve standardized sampling and multi-point comprehensive evaluation, and thus obtain a stable and reliable individual shell hardness phenotype has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] For the reasons stated above, the purpose of this invention is to provide a standardized process for measuring and integrating the phenotypic characteristics of oyster shell hardness. Through multi-site sampling and mean-based data processing, differences at different locations and between the left and right shells are offset, achieving accurate characterization of the overall mechanical properties of the oyster. This method specifically includes the following steps:

[0006] Sample pretreatment: The shells of the Pacific oyster were cleaned at room temperature to remove surface deposits and avoid differences in shell hardness.

[0007] Standardization of measurement sites: To reduce the differences in irregular oyster shell morphology, the longest axis (maximum length) and the widest axis (maximum width) were determined in the contours of the left and right shells, respectively. An orthogonal reference cross axis was constructed at the intersection of the two axes. Based on this, three standardized measurement sites were set at a position 5 mm inside the shell edge along this axis and marked with circular markers.

[0008] Sample preparation: Use a cutting tool to cut the shell at the marked positions on the left and right shells to obtain 6 flat shell samples;

[0009] Multi-point microhardness measurement: Six shell samples of each individual were measured sequentially using a Vickers microhardness tester. A preset 10 gf micro-load was applied to each marked area, held for approximately 10 seconds, and five indentation tests were performed within each marked area to obtain the original hardness value of that area.

[0010] Data cleaning and site hardness value calculation: Abnormal hardness measurements caused by internal micro-defects and other reasons are removed from the original hardness values. The arithmetic mean of the remaining valid measurements is calculated as the representative hardness value of each shell sample.

[0011] Construction of Individual Comprehensive Hardness Index: Representative hardness values ​​from six shell samples were comprehensively calculated to obtain a comprehensive hardness index characterizing the overall mechanical properties of the individual shell. As a key support for the scientific validity and effectiveness of the method of this invention, the method also includes a complete feasibility verification process:

[0012] By using nonparametric tests and homogeneity of variance tests, the inherent micromechanical heterogeneity of the shell surface is quantitatively confirmed, thus demonstrating the limitations of single-point measurement and the necessity of using multi-point measurement in this invention.

[0013] Bivalve consistency analysis and data integration rationality verification: The systematic differences in hardness between the left and right shells were compared by examining paired samples, and their correlation was calculated. If the hardness of the left and right shells showed a moderate to high significant positive correlation and homogeneous variances, it proved that the bivalve can be used as repeated observations of the same genetic potential, providing a core basis for integrating left and right shell data into a single individual phenotype.

[0014] Phenotypic Variance Decomposition and Signal Evaluation: Using a linear mixed-effects model, the total phenotypic variance is decomposed into between-individual variance and residual variance. The contribution rate of between-individual variance is calculated, which directly reflects the stable signal strength of the phenotypic data.

[0015] Population phenotypic distribution test: Apply the method of this invention to a large population to test the distribution characteristics of the obtained individual comprehensive hardness index. It should show a continuous distribution and be approximately normal, which is consistent with the quantitative trait characteristics controlled by multiple genes.

[0016] The beneficial effects of this invention are as follows:

[0017] Compared with existing technologies, this invention has significant advantages. By standardizing the selection of measurement sites and employing multi-point sampling tests for microhardness, this invention effectively overcomes the inherent spatial heterogeneity of the microstructure in the shells of the Pacific oyster, significantly reducing random fluctuations in measurement results and making the obtained individual shell hardness phenotypic data more stable and reliable, with a significantly improved signal-to-noise ratio. Simultaneously, this invention establishes a clear and unified shell location calibration method, explicit spatial distance limitations, and a standardized measurement process, thereby ensuring good repeatability and consistency of measurement results under different operators and laboratory conditions. Furthermore, this invention innovatively integrates a complete scientific verification system from shell heterogeneity confirmation and data integration rationality proof to signal intensity quantification, providing a systematic decision chain and statistical evidence for the conversion of micromechanical measurement results into usable phenotypes for genetic breeding, demonstrating outstanding innovation and application promotion value. Attached Figure Description

[0018] Figure 1 is a schematic diagram showing the marking of 6 standardized measurement sites on the left and right shells of the Pacific oyster.

[0019] Figure 2 shows the differences and distribution of hardness values ​​at different measurement sites on the shell of the Pacific oyster.

[0020] Figure 3 is a box plot comparing the average hardness of the left and right shells.

[0021] Figure 4 is a scatter plot showing the correlation and consistency of the hardness values ​​of the left and right shells.

[0022] Figure 5 is a histogram showing the frequency distribution of the overall shell hardness values ​​of the Pacific oyster population.

[0023] Figure 6 is a box plot comparing the overall shell hardness of different source groups (Haida 1 and Haida 4). Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0025] The first embodiment of the present invention is a standardized measurement method for the hardness of oyster shells, comprising the following steps:

[0026] Intact oysters were selected, and their shells were cleaned to remove surface impurities, avoiding high-temperature treatment during the cleaning process. Six measurement areas were then selected along the axis of the left and right shells, 5 mm from the shell edge, and marked with 6 mm diameter circular markers (Figure 1). Measurement areas on the left shell were numbered 1-3, and those on the right shell were numbered 4-6. Shell fragments were cut along the marked areas using a small electric saw, and the sample blocks were placed on the stage of an HVS-1000AT micro Vickers hardness tester. A load of 10 gf and a holding time of 10 seconds were selected. A flat area without obvious defects was selected within each marked circle, and five indentation tests were performed consecutively. The instrument automatically recorded the Vickers hardness value for each test and calculated the average value as the microhardness value for that area. Finally, the total average hardness of the six test areas was calculated and used as the shell hardness index for this oyster individual for subsequent population comparison and genetic analysis.

[0027] In a preferred embodiment, to verify the stability, statistical rationality, and genetic analysis applicability of the above measurement method under large sample conditions, a certain scale of Pacific oyster individuals were selected for systematic verification.

[0028] Feasibility verification and effect data of the embodiments:

[0029] In the preferred embodiment described above, 420 individual Pacific oysters were selected, and microhardness was measured at six standard measurement sites on the left and right shells of each individual according to the standardized measurement method described in this invention. The obtained phenotypic data were then systematically evaluated as follows:

[0030] Heterogeneity confirmation: Measurements at six standard sites in 420 oyster individuals showed highly significant differences in hardness among sites (Kruskal-Wallis test, P<0.001) and unequal variances (Levene test, P<0.001), confirming the existence of micromechanical heterogeneity in the shells. This, in turn, demonstrates the inadequacy of single-point measurements and the necessity of multi-point sampling in this invention. Figure 2 ).

[0031] The data integration demonstrates its rationality: Although there are systematic differences in the hardness of the left and right shells (paired t-test, P<0.001), their variances are homogeneous (F-test, P = 0.140) and they exhibit a moderately significant positive correlation (Pearson's r = 0.592, P<0.001). This correlation proves that the left and right shells are repeated measurements of the hardness of the same whole shell, satisfying the basic statistical premise for data integration (Figure 3-4).

[0032] Quantification of genetic signal intensity: Variance component analysis was performed using a linear mixed-effects model. The results showed that the variance among individuals (σ...)2 individual The residual variance (σ) is 102.75. 2 residual The variance was 77.93. The inter-individual variance accounted for 56.9% of the total phenotypic variance. This key indicator demonstrates that the variation in the individual composite hardness index obtained through the method of this invention is mainly (more than half) due to stable genetic or physiological differences between individuals, rather than measurement noise or local random variation. This provides strong statistical support for using this phenotype in genome-wide association studies (GWAS).

[0033] Population Distribution Characteristics: Statistical analysis was performed on the comprehensive shell hardness values ​​of 420 oyster individuals from two different populations (Haida No. 1 and Haida No. 4). The results showed that the index exhibited a continuous and approximately normal distribution, ranging from 60 to 120 HV, indicating that the shell hardness phenotype obtained by the method of this invention has good continuity and discriminative ability, and is suitable for population-level individual difference analysis (Figure 5). Based on this distribution characteristic, individuals with high shell hardness at the high end of the distribution (e.g., the upper quantile interval) can be defined as having excellent mechanical properties, possessing potential advantages in resisting physical impact, biological erosion, and adverse environmental stress, and are suitable as candidate parents for stress resistance selection and genetic improvement. Further comparative analysis of oysters from different populations revealed that the shell hardness of the Haida No. 1 population, after 17 generations of selective breeding, was more stable, and there was a statistically significant difference in the comprehensive shell hardness level between it and the Haida No. 4 population from the same sea area (Welch's test, P < 0.0001), indicating a detectable systematic difference in shell mechanical properties among different population sources. Figure 6 The method of this invention can reliably distinguish the differences in hardness among the aforementioned groups, indicating that it has good sensitivity and discriminative ability to real biological variations.

[0034] The above results indicate that the standardized method for measuring the microhardness of oyster shells provided by this invention has good statistical reliability and representativeness, and is suitable for applications such as large-sample phenotypic determination and genetic association analysis.

[0035] The above description is merely a preferred embodiment of the present invention and does not limit the scope of the patent. Any equivalent structural transformations made based on the inventive concept of the present invention and the description and drawings, or direct / indirect applications to other related technical fields, should be included within the scope of patent protection of the present invention.

Claims

1. A method for evaluating the hardness of oyster shells, characterized in that, Includes the following steps: (1) Sample pretreatment: Clean the double shells of the Pacific oyster at room temperature to remove surface attachments and avoid differences in shell hardness; (2) Standardization of measurement sites: In order to reduce the difference in irregular oyster shell shape, the longest axis (maximum length) and the widest axis (maximum width) were determined in the contours of the left and right shells respectively, and an orthogonal reference cross axis was constructed at the intersection of the two. Based on this, three standardized measurement sites were set at a position 5 mm inside the shell edge along this axis and marked with circular marks. (3) Sample preparation: Use a cutting tool to cut the shell at the left and right shell markings to obtain 6 flat shell samples; (4) Multi-point measurement of microhardness: Six shell samples of each individual to be tested were measured sequentially using a Vickers microhardness tester. A preset micro-load of 10 gf was applied to each marked area and held for about 10 seconds. Five indentation tests were performed in each marked area to obtain the original hardness value of that area. (5) Data cleaning and site hardness value calculation: Remove abnormal hardness measurement values ​​caused by internal micro-defects and other reasons from the original hardness values, and calculate the arithmetic mean of the remaining valid measurement values ​​as the representative hardness value of each shell sample. (6) Construction of individual comprehensive hardness index: The representative hardness values ​​of 6 shell samples were comprehensively calculated to obtain the comprehensive hardness index that characterizes the overall mechanical properties of the individual shell.

2. The method according to claim 1, characterized in that, In step S2, the standard measurement point is set at a distance of about 5 mm from the edge of the shell; the number of measurement points is three for each of the left and right shells; the measurement point is located using a circular area with a diameter of about 6 mm, which can accurately characterize the overall mechanical properties of the shell by measuring the comprehensive hardness index.

3. The method according to claim 1, characterized in that, In step S4, the small load of 10 gf and the holding time of about 10 seconds ensure that the indentation size is moderate and the boundary is clear, thus guaranteeing the interpretability and consistency of the test results; avoiding excessive damage to the nascent shell structure; the sampling design of performing 5 indentation tests in each marked area can effectively reduce random errors, improve the stability and repeatability of the measurement results, and thus obtain more accurate shell hardness data for each measurement point.

4. The method according to claim 1, characterized in that, In step S6, the individual comprehensive shell hardness index is obtained by averaging the microhardness values ​​of six measurement sites on the left and right shells. This reduces the random measurement error caused by the inherent micromechanical heterogeneity of the shell surface and reduces the random deviation caused by the measurement of a single site, thereby more accurately and stably characterizing the overall mechanical properties of the oyster shell.

5. The method according to claim 1, characterized in that, The shell hardness value measured by the above method can be used as an indicator to assess the oyster's stress resistance and the degree of sea acidification, assisting in environmental monitoring and aquaculture production.

6. The method according to claim 1, characterized in that, The above-mentioned measurement method can accurately quantify the shell hardness index, enabling selective breeding of oysters with high shell hardness, cultivating superior varieties that are resistant to stress and acidification and have strong biological carbon sequestration, thus providing value to the industry.

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