Ruditapes philippinarum blood cell function classification method based on ultrastructure change
By setting different dissolved oxygen changes modes and observing the ultrastructure changes of blood cells, the problem of functional classification of shellfish blood cells was solved, and the accurate functional classification and active state of Filipino clam blood cells was achieved.
Patent Information
- Application Number
- CN202411877918.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-25
AI Technical Summary
The prior art is difficult to effectively classify and study the functions of shellfish blood cells, especially in response to dissolved oxygen changes, and lacks a unified blood detection method.
Using a method based on ultrastructural changes, the Filipino clams were treated with dissolved oxygen by setting different dissolved oxygen changes modes, and then the ultrastructural changes of blood cells were observed through scanning electron microscopy and transmission electron microscopy, and functional classification was performed.
The accurate functional classification of Filipino clam blood cells was achieved, the small individual limitations were overcome, the natural dissolved oxygen changes were simulated, and the active state of blood cells was preserved to the greatest extent. It was suitable for repeated experiments and comparison experiments.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of marine biotechnology, and specifically relates to a blood cell function classification method of Ruditapes philippinarum based on ultrastructural changes. Background Art
[0002] A large number of studies have shown that shellfish do not have a specific immune system and cannot produce antibodies in response to changes in the external environment. However, as an invertebrate with a relatively primitive classification status, shellfish also have an effective immune defense method to regulate the body's adaptation to environmental changes during the long process of evolution. The phagocytosis, encapsulation and production of reactive oxygen molecules of their blood cells play this role (Li Taiwu et al., Blood cells of Abalone variegated, Journal of Fisheries of China, 2007, 31 (Suppl): 12-17).
[0003] Blood (lymph) fluid has four main functions in animal life activities: transportation, defense, participation in body fluid regulation and maintaining internal environment homeostasis. The hemolymph of bivalve shellfish is closely related to environmental factors, the physiological state of the animal itself, and pathogenic microorganisms (Beatrice Gagnaire, Patrick Soletchnik, Nicole Faury, et al. Analysis of hemocyte parameters in Pacific oysters, Crassostrea gigas, realed in the field—Comparison of hatchery diploids and diploids from natural beds.
[0004] Aquaculture, 2007, 264: 449-456). In addition to immune functions, blood cells suspended in the hemolymph of bivalve shellfish also participate in other physiological functions. For example, blood cells between the mantle cavity and the shell participate in biomineralization and shell growth; blood cells covered in the mucus layer on the body surface have the function of sentinel warning; blood cells also have the functions of wrapping larger invading foreign bodies, participating in wound healing, transporting nutrients, helping food digestion, and secreting body fluid factors (Lau Yuk-Ting, Sussman Lauren, Espinosa Emmanuelle Pales, et al. Characterization of hemocytes from different body fluids of the eastern oyster Crassostrea virginica. Fish and Shellfish Immunology, 2017, 71: 372-379). However, there are not many studies on the transport of substances (especially O2), metabolism, and response to adverse environmental factors of shellfish blood cells, and most of them are derived from related studies on vertebrates, which may lead to cognitive errors.
[0005] Bivalve molluscs are rich in species and their habitats vary greatly. The functional classification of blood cells in different species may be different. At present, the classification of shellfish blood cells is roughly divided into two categories: granular cells and transparent cells (no or few granules in the cytoplasm) according to whether the cells contain granules. There are also many intermediate cells between the two major types of cells. There are few research reports on the functional classification of blood cells. Since no substantial evidence can be provided, the functional classification description is mostly inferential. Only two papers mentioned the function of granules in granulocytes. For example, the cytoplasm of granulocytes of geoduck (Panopea globosa) contains a large number of basophilic granules, and the round nucleus is off-center; while the cytoplasm of transparent cells is eosinophilic (LS Hernández-Méndez, E. Castro-Longoria, C. L. Araujo-Palomares, et al. Hemocyte cell types of the cortesgeoduck, Panopea globosa (Dall 1898), from the Gulf of California, Mexico. Fish and Shellfish Immunology, 2020, 100: 230–237); Seiler and Morse (Gregory R. Seiler, M. Patricia Morse. Kidney and Hemocytes of Mya arenaria (Bivalvia): normal and pollution-Related ultrastructural morphologies. Journal of Invertebrate Pathology, 1988, 52: 201-214) also confirmed that the granules in the blood cells of the sand sea beetle (Mya arenaria) are different types of lysosomes at different stages, containing hydrolases such as acid phosphatase, alkaline phosphatase or lipase. These granulocytes have the function of removing and degrading pollutants in the body.Boisseaux et al. (Paul Boisseaux, Marie-Laure Delignette-Muller, Khedidja Abbaci, et al. Analysis of hemocytes in Lymnaea gnalis: Characterization and effects of repeated hemolymph collections. Fisher and Shellfish Immunology, 2016, 57: 116-126) believed that mollusk blood cells are highly plastic and free-drifting cells, and the differences between them may be due to different cell maturity, physiological state and / or in vitro state before fixation.Moreover, different analysis methods lead to very different results in the identification of blood cell functions. For example, using ordinary optical microscope and flow cytometer to observe and analyze, the blood cells of long oysters (Crassostrea gigas) are divided into three types: non-granulocytes (agranulocytes), clear cells (hyalinocytes), and granulocytes (granulocytes). Under hypoxic conditions, the number of non-granulocytes increases, while the number of granulocytes and clear cells decreases. However, except for the obvious phagocytic function of granulocytes, the functions of non-granulocytes and clear cells are still unclear. It is believed that 24h hypoxia will lead to a decrease in blood cell ROS, but will not lead to blood cell death (Aleksandra Yu. Andreyeva, Ekaterina S. Kladchenko, Oksana Y. Vyalova, et al. Functional characterization of the Pacific oyster, Crassostrea gigas (Bivalvia: Ostreidae), hemocytes under normoxia and short-term hypoxia. Turkish Journal of Fisheries & Aquatic Science.2021, 21(3):125-133); Under electron microscopic observation, the functional classification of blood cells in Scapharca broughtonii is more clear, divided into red blood cells (RBCs) with hemoglobin granules, white blood cells (WBCs) with phagocytic function, and thrombocytes (TCs) with coagulation function (Liqing Zhou, Dan Zhao, Biao Wu, et al. Ark shell Scapharca broughtonii hemocyte response against Vibrio anguillarum challenge. Fish and Shellfish Immunology, 2019, 84:304–311). Blood cells respond to the recognition and destruction of non-self factors, so blood cells are considered to be important participants in the immune response of shellfish. Their total number and morphological and functional characteristics are key parameters for assessing the health of organisms and are also necessary to understand their role in shellfish immune defense.
[0006] Most species of bivalve molluscs are filter feeders, which have a good purification and repair effect on coastal tidal flat ecosystems and are an important part of marine ecosystems. Bivalve mollusc farming has a long history in my country's marine aquaculture industry, and the farming technology is also very mature. In the increasingly intensive farming environment, farmed shellfish are more susceptible to various adverse environmental factors and increase the risk of disease outbreaks. In recent years, global climate change and eutrophication of coastal seawater have caused the low oxygen zone of the tidal flat ecosystem to continue to expand. The severity and duration of hypoxia will be aggravated by eutrophication and may worsen with climate change. Whether long-term or short-term, rapid or slow, changes in dissolved oxygen will have an adverse effect on the growth and development of shellfish. Observation of the structure and function of blood cells can not only be used to evaluate the physiological state of shellfish, but also serve as a valuable tool to monitor the impact of environmental changes on animal health. Mammals and fish have established relatively mature blood routine detection methods, but there is no unified standard blood detection method for invertebrates such as shellfish. Summary of the invention
[0007] The technical problem to be solved by the present invention is to provide a method for classifying the function of blood cells of Philippine clams based on ultrastructural changes, the method is a method for classifying the function of blood cells according to the ultrastructural change characteristics, the method is to set 3 dissolved oxygen change modes, namely, to maintain normal dissolved oxygen (C) treatment, normal dissolved oxygen-rapid hypoxia-rapid reoxygenation (AHR) treatment, and normal dissolved oxygen-slow hypoxia-slow reoxygenation (CHR) treatment, and to treat a certain amount of Philippine clams with dissolved oxygen changes. Then, 3-6 Philippine clams hemolymph fluids are randomly extracted according to the size of the clams, and blood cell scanning and transmission electron microscopy are performed after fixation, and then functional classification is performed according to the ultrastructural changes of blood cells. The method of the present invention can overcome the limitation that the individual of Philippine clams is relatively small, simulate various dissolved oxygen changes in natural waters, and preserve the active state of blood cells in response to environmental changes and their normal reaction morphology in the body to the greatest extent, and the operation is simple and convenient, which can meet the needs of repeated experiments and comparative experiments, and accurately realize the functional classification of blood cells of Philippine clams.
[0008] The present invention is accomplished according to the following operating method:
[0009] A method for classifying blood cell functions of Manila clams based on ultrastructural changes is provided. The method comprises the following steps: setting three dissolved oxygen change modes for cultured Manila clams, which are: maintaining a normal dissolved oxygen of 7.0 mg / L all the time; rapid hypoxia 24 hours-rapid reoxygenation 4 hours treatment: firstly controlling the dissolved oxygen to decrease from 7.0 mg / L to 0.5 mg / L within 4 hours by nitrogen filling method, starting timing, applying hypoxia stress for 24 hours, and then immediately aerating to increase the dissolved oxygen from 0.5 mg / L to 7 mg / L within 4 hours, i.e., rapid reoxygenation 4 hours; and slow hypoxia 48 hours-slow reoxygenation 8 hours treatment: firstly controlling the dissolved oxygen to decrease slowly to 0.5 mg / L within 8 hours by nitrogen filling method, then continuing the hypoxia stress for 48 hours, and then gradually aerating to increase the dissolved oxygen slowly to 7 mg / L within 8 hours, and the reoxygenation process lasts for 8 hours. Normal dissolved oxygen treatment was used as the control group. Three replicates were set up for each of the three models, and the same number of Manila clams were evenly placed in each replicate. Dissolved oxygen was measured and mortality statistics were performed every 2 hours. No feeding or water changes were performed during the treatment period.
[0010] As a preferred embodiment, the Manila clams were not fed 24 hours before the experiment.
[0011] Blood was randomly collected from 3-6 Philippine clams in each treatment group, and blood was quickly drawn twice from the blood sinus of the posterior adductor muscle of each Philippine clam. Blood was drawn from the normal dissolved oxygen group 24 hours after treatment, serving as the control group. Blood was drawn from the rapid hypoxia 24 hours-rapid reoxygenation 4 hours treatment group at two time points: 24 hours after rapid hypoxia stress and 4 hours after rapid reoxygenation. Blood was drawn from the slow hypoxia 48 hours-slow reoxygenation 8 hours treatment group at two time points: 48 hours after slow hypoxia and 8 hours after slow reoxygenation. The twice-collected blood from each treatment group was directly and quickly mixed into a container pre-filled with electron microscope fixative, which was placed on ice and finally placed at 4°C for standing. After blood cells were deposited at the bottom of the test tube, the supernatant was drawn and discarded, the fixative was replaced once, and the blood was stored at 4°C for later use.
[0012] As a preferred embodiment, the electron microscopy fixative contains 4% paraformaldehyde by volume, 2.5% glutaraldehyde by volume and sterile filtered seawater with a molar concentration of 0.05M Tris, with a pH of 7.2.
[0013] Then, the fixed samples were subjected to scanning electron microscopy and transmission electron microscopy sample preparation, respectively, and then observed under scanning electron microscopy and transmission electron microscopy, respectively. The blood cells were functionally classified according to the cell morphological changes observed under scanning electron microscopy and the ultrastructural changes in the blood cells, including granules, mitochondria, endoplasmic reticulum, cell nucleus and Golgi apparatus, observed under transmission electron microscopy. The specific classification standards are morphological standards and ultrastructural standards. The morphological standards are as follows: (1) the cell surface extends with sheet-like or warty pseudopodia, which can move toward foreign bodies or wrap and engulf foreign bodies. When the dissolved oxygen changes, the cell pseudopodia have strange shapes and the mobility becomes weak. Such cells are deformed cells.
[0014] (2) They always maintain a spherical shape, with small openings for exocytosis of secretory vesicles or secretions adhering to the cell surface. When the dissolved oxygen changes, the amount and state of secretions on the cell surface will change. These cells are secretory cells.
[0015] (3) In the normal dissolved oxygen group, the cells are spindle-shaped with one end thick and the other end thin, and the nucleus is large and rugby-shaped. When the dissolved oxygen changes, the thrombocytes will undergo morphological changes, affecting the coagulation function. These cells are thrombocytes;
[0016] (4) The normal morphology is spherical, and the cell surface is relatively flat. However, when the dissolved oxygen changes, the cell surface will collapse or bulge, and the spherical cells will become irregular. These cells are spherical cells.
[0017] (5) The cells are generally spherical, and the cell membrane is wrinkled. The wrinkles are large but regular and symmetrical. When the dissolved oxygen changes, the wrinkles become messy and irregular due to changes in surface tension, and many depressions appear on the cell surface, or the cells lose their spherical structure. There are pinocytic vacuoles or phagocytic vacuoles in the cells. These cells are macrophage-like cells.
[0018] The ultrastructural criteria are:
[0019] (1) The nucleus is round and centered. The granules in the cytoplasm are irregular in size and shape, with a diameter of 0.2-0.4 μm, and are scattered in the cytoplasm. The mitochondria are unevenly distributed or clustered together, mostly in an oblong shape. The endoplasmic reticulum near the nucleus is abundant, the smooth endoplasmic reticulum is round and vesicular, and the rough endoplasmic reticulum is elongated and tubular. The boundaries between phagocytic vacuoles and pinocytic vacuoles are clear, the phagocytic vacuoles contain granular inclusions, and the pinocytic vacuoles are empty or contain solution. The cells can be seen engulfing foreign bodies, and there are Golgi bodies in the cytoplasm. Within 24 hours Rapid hypoxia has little effect on the ultrastructure of cells, but after rapid reoxygenation, cells lose the ability to deform and move. The pseudopodia have poor membrane fluidity and their ability to engulf foreign matter becomes weaker. The outer membrane of particles in the cytoplasm falls off and becomes loose. The outer membrane of mitochondria is damaged, or the double membrane cannot be observed. Some cristae fall off or are arranged irregularly, and phagocytic vacuoles rupture. The number of endoplasmic reticulum vesicles decreases, and the morphology of undamaged endoplasmic reticulum vesicles changes. There are gaps in the cytoplasm, and cells undergo apoptosis. These cells are deformed cells.
[0020] (2) The nucleus is large, spherical, and off-center; the granules in the cytoplasm are large and numerous, evenly distributed in the cytoplasm, with a diameter of 0.45-0.55 μm. There are 13-16 granules in a cell section, which are spherical or crescent-shaped, accompanied by secretory vesicles; the mitochondria are oval, with uniform size distribution; the secretory vesicles are also evenly distributed in the cell, containing abundant fine granular secretions, and secretions are seen on the cell surface; the endoplasmic reticulum is abundant, with rough and smooth endoplasmic reticulum interwoven, the smooth endoplasmic reticulum is vesicular, and the rough endoplasmic reticulum is tubular; rapid hypoxia stress Under the condition of 40 ℃ and 80 ℃, the mitochondrial double membrane is difficult to distinguish, and the cristae of most mitochondria fall off; the endoplasmic reticulum membrane vesicles are unevenly distributed in the cytoplasm, and more than 90% of the endoplasmic reticulum is rough endoplasmic reticulum; the outer membrane of the granules falls off, and the shape becomes irregular; some secretory vesicles surrounded by membrane vesicles rupture or fuse with each other, causing cell apoptosis; after slow reoxygenation, the hypoxic damage of the cells is more obvious, the mitochondrial double membrane is not clear, the cristae also fall off, the granules are irregular in shape, and the contents of the secretory vesicles surrounded by membrane vesicles are small and unevenly arranged. These cells are secretory cells;
[0021] (3) The nucleus is large and long, and the cytoplasm is rich in endoplasmic reticulum and Golgi membrane vesicles. More than 90% of the endoplasmic reticulum is tubular rough endoplasmic reticulum. The mitochondria are clustered together. There are unique double-membrane vesicles in the cells, which are either empty or contain solution. There are no particles in the cytoplasm. These cells are thrombocytes. Under rapid hypoxia stress, the cristae of most mitochondria in thrombotic cells fall off, the double-membrane structure is incomplete, the mitochondria are irregular in shape, and the endoplasmic reticulum membrane vesicles do not undergo obvious changes.
[0022] (4) The cell nucleus is spherical and deviates from the center. The mitochondria in the cytoplasm are very abundant, more than 90% of which are long strips and easy to aggregate. The endoplasmic reticulum is scarce. There are 0-3 small clusters between the nucleus and the cell membrane in the cell section, most of which are slender tubular. More than 90% of the endoplasmic reticulum is smooth. There are Golgi bodies in the cytoplasm. The particles in the cytoplasm are mostly regular spherical, with a diameter of 0.35-0.45μm. Under normal dissolved oxygen conditions, there are particles in the cell section, but the number and shape of the particles change with the change of dissolved oxygen. Under the change of dissolved oxygen, the damage caused by hypoxia is prominent, the structure of the mitochondrial double membrane and cristae are incomplete, the boundaries of the particles are not clear, the shape is elongated into an oblong shape, and even diffuse disintegration occurs. The number increases to 9-11, but the shapes are different. The number of endoplasmic reticulum membrane vesicles is even scarcer, and they are sparsely aggregated into 1-3 piles. This type of cell is a spherical cell.
[0023] (5) The nucleus is large, irregular in shape, or lobed, with a high nuclear-cytoplasm ratio; the cell membrane is wrinkled to form vacuoles, the rough endoplasmic reticulum is abundant in the cell, almost filling the entire cytoplasm, ribosome particles are attached to the elongated tubules, and there are also a large number of free ribosome particles in the cytoplasm; the mitochondria are long ovals of uniform size and evenly distributed in the cell; there are no particles in the cytoplasm; regardless of normoxia, hypoxia or reoxygenation, phagocytic vacuoles or pinocytic vacuoles are found in the cells, and these cells are macrophages.
[0024] As a preferred embodiment, scanning electron microscope sample preparation and observation: use 0.1M pH7.4 phosphate buffer PBS to rinse the fixed sample 3 times, each time for 15 minutes, then add 0.1M phosphate buffer PBS containing 1% by mass osmium acid and fix it at room temperature in the dark for 1-2 hours, and rinse it again with 0.1M pH7.4 phosphate buffer PBS for 3 times, each time for 15 minutes; then gradient dehydration: blood cells are sequentially placed in →30%→50%→70%→80%→90%→95%→100%→100% alcohol, each time for 15 minutes; then placed in pure isoamyl acetate for 15 minutes; finally, the sample is placed in a critical point dryer for drying and sprayed with gold on an ion sputtering instrument for 30 seconds, and then placed in a scanning electron microscope for observation and image collection.
[0025] As a preferred embodiment, transmission electron microscopy sample preparation and observation: the fixed sample is fixed with 1% osmium acid prepared in 0.1M pH 7.4 phosphate buffer PBS in the dark for 2 hours at room temperature, and then rinsed with 0.1M pH 7.4 phosphate buffer PBS 3 times, each time for 15 minutes. Then dehydrate in a gradient at room temperature: blood cells are sequentially dehydrated in 30% → 50% → 70% → 80% → 95% → 100% → 100% alcohol, each time for 20 minutes, and dehydrated twice with 100% acetone, each time for 15 minutes; then infiltration embedding, that is, the volume ratio of acetone: 812 embedding agent is 1:1, and react at 37°C for 2-4 hours; the volume ratio of acetone: 812 embedding agent is 1:2, and infiltration is carried out at 37°C overnight; pure 812 embedding agent is embedded at 37°C for 5-8 hours; then pure 812 embedding agent is poured into the embedding plate, and the sample is placed in the embedding plate and incubated at 37°C overnight; then embedding The embedded plate was placed at 60℃ for polymerization for 48h, and the resin block with the embedded sample was taken out for use; then ultrathin sectioning: the resin block was sliced into 60-80nm ultrathin sections on an ultrathin section machine, and the sections were scooped out on a 150-mesh square copper mesh; section staining: the copper mesh was stained with a 2% by volume uranyl acetate saturated alcohol solution in the dark for 8min; washed 3 times with 70% by volume alcohol; washed 3 times with ultrapure water; stained with a 2.6% by mass lead citrate solution in the dark for 8min; washed 3 times with ultrapure water, and slightly dried with filter paper; the copper mesh sections were placed in a copper mesh box and dried overnight at room temperature; finally, the sections were observed under a transmission electron microscope, and image analysis was collected.
[0026] The beneficial effects of the present invention compared with the prior art are as follows:
[0027] The present invention sets three dissolved oxygen change modes by simulating various dissolved oxygen changes in natural sea areas, and then based on the ultrastructural changes of blood cells, clearly distinguishes according to morphological characteristics, without intermediate forms and ambiguous forms, and accurately classifies the functions of blood cells of the Philippine clam Manila clam, which can overcome the limitation of small experimental shellfish individuals. The method can preserve the active state of blood cells in response to environmental changes and their normal reaction morphology in the body to the greatest extent, and can meet the needs of repeated experiments and comparative experiments, thereby realizing the accurate classification of the blood cell functions of the Philippine clam Manila clam.
[0028] The method of the present invention consists of three steps: setting of dissolved oxygen mode, collection and fixation of blood cells, and observation of blood cell ultrastructure and functional classification. It is a systematic method, and none of the three steps can be missing. The blood cells of the Philippine clam obtained by the method basically maintain their active form in the body, and the types of blood cells taken are comprehensive. After rapid fixation, the morphology and function of the blood cells are instantly fixed, ensuring the accuracy of subsequent observation and functional classification. It is particularly suitable for observing the morphological changes of blood cells of various bivalve molluscs and their functional analysis, and is also expected to develop into a routine blood detection method for bivalve molluscs. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 Effects of dissolved oxygen changes on the morphology of five blood cells (scale: 5 μm); a. Morphology of amebocytes in normoxia, f. Morphology of amebocytes after rapid hypoxia for 24 hours, k. Morphology of amebocytes after rapid reoxygenation for 4 hours, p. Morphology of amebocytes after slow hypoxia for 48 hours, u. Morphology of amebocytes after slow reoxygenation for 8 hours;
[0030] b. Morphology of secretory cells in normoxia, g. Morphology of secretory cells in rapid hypoxia for 24 hours, l. Morphology of secretory cells in rapid reoxygenation for 4 hours, q. Morphology of secretory cells in slow hypoxia for 48 hours, v. Morphology of secretory cells in slow reoxygenation for 8 hours;
[0031] c. morphology of thrombus cells in normoxia, h. morphology of thrombus cells after rapid hypoxia for 24 hours, m. morphology of thrombus cells after rapid reoxygenation for 4 hours, r. morphology of thrombus cells after slow hypoxia for 48 hours, w. morphology of thrombus cells after slow reoxygenation for 8 hours;
[0032] d. morphology of spheroid cells under normoxia, i. morphology of spheroid cells after rapid hypoxia for 24 h, n. morphology of spheroid cells after rapid reoxygenation for 4 h, s. morphology of spheroid cells after slow hypoxia for 48 h, x. morphology of spheroid cells after slow reoxygenation for 8 h;
[0033] e. Morphology of macrophage-like cells in normoxia, j. Morphology of macrophage-like cells in rapid hypoxia for 24 hours, o. Morphology of macrophage-like cells in rapid reoxygenation for 4 hours, t. Morphology of macrophage-like cells in slow hypoxia for 48 hours, y. Morphology of macrophage-like cells in slow reoxygenation for 8 hours;
[0034] Figure 25 Ultrastructural morphological characteristics of blood cells under transmission electron microscope;
[0035] a. Ultrastructural morphology of deformed cells (scale ruler: 2 μm), f. Ultrastructural morphology of deformed cells (scale ruler: 1 μm); b. Ultrastructural morphology of secretory cells (scale ruler: 2 μm), g. Ultrastructural morphology of secretory cells (scale ruler: 1 μm); c. Ultrastructural morphology of thrombocytes (scale ruler: 2 μm), h. Ultrastructural morphology of thrombocytes (scale ruler: 1 μm); d. Ultrastructural morphology of spherical cells (scale ruler: 2 μm), i. Ultrastructural morphology of spherical cells (scale ruler: 1 μm); e. Ultrastructural morphology of macrophage-like cells (scale ruler: 2 μm), j. Ultrastructural morphology of macrophage-like cells (scale ruler: 1 μm);
[0036] ER: endoplasmic reticulum, rER: rough endoplasmic reticulum, sER: smooth endoplasmic reticulum, GA: Golgi apparatus, G: granule, MT: mitochondria, N: nucleus, PHV: phagocytic vesicle, PIV: pinocytotic vesicle, Ss: secretions, SEVs: secretory vesicles, The red arrow indicates the unique double-layer membrane bubble. ) 。 DETAILED DESCRIPTION
[0037] The present invention is described in detail below through specific embodiments in conjunction with the accompanying drawings. The following is a specific operation process of the best embodiment, and the protection scope of the present invention is not limited in any form by the embodiments.
[0038] Example 1
[0039] The Philippine clam is an important economic bivalve mollusc widely distributed throughout the world. There have been some reports on the classification and ultrastructural observation of its blood cells. Wang Wenqi et al. (Wang Wenqi, Xu Shenbo, Zhang Yuna, et al. A preliminary study on the classification and function of blood cells of the Philippine clam (Ruditapes philippinarum). Journal of Laiyang Agricultural College (Natural Science Edition), 200623(3):165-169) used histochemical methods to classify the blood cells of the Philippine clam into four categories: transparent blood cells, small granular blood cells, large granular blood cells and lymphoid blood cells according to the presence or absence of morphological and structural characteristic particles, and Liu Dongwu et al. (Liu Dongwu, Wang Yiyan, Sun Hushan. Classification of blood cells of the Philippine clam, Chinese clam, clam and purple stone clam. Fisheries Science , 2005, 24(10): 5-7), Ren Xingchao et al. (Ren Xingchao, Li Rong, Zhang Ning, et al. Image flow cytometry classification analysis of blood cells of Meretrix meretrix and Manila clams. Anhui Agricultural Sciences, 2020, 48(8): 98-101) observed the classification results using flow cytometry and the results were consistent; Liu Jing (Liu Jing, Yuan Yuqing, Wu Yalin. Preliminary study on the preparation of transmission electron microscopy samples of blood cells of Manila clams. Bulletin of Oceanology and Lakes, 2016, (6): 113-119) discussed the method of preparing transmission electron microscopy samples of blood cells of Manila clams, but there have been no reports on the ultrastructure of blood cells of Manila clams in response to changes in dissolved oxygen. The standard for blood cell classification based solely on the presence or absence of granules in the cytoplasm is still unclear, and the specific functions of each type of cell cannot be determined.
[0040] On April 20, 2021, about 500 6kg 1-2 year old Manila clams of uniform size were collected from Hongdao Beach, Qingdao City, Shandong Province. The shell length was 36.49±2.72mm, the shell height was 25.45±2.65mm, the shell width was 15.71±1.24mm, and the body weight was 9.13±2.04g. After the clams were collected, they were temporarily kept in the laboratory for about a week for experiments. The temporary water temperature was controlled at 19-21℃. During the temporary period, diatoms, flat algae and golden algae cultivated in the laboratory were fed (1L fresh algae solution was fed to 20L water). The water was changed once a day, and half of the seawater was changed each time, and the residual bait and feces were sucked out once. 30L plastic tanks without bottom sediment were used for both experiments and temporary culture. Stop feeding unicellular algae 24h before the experiment.
[0041] (1) Dissolved oxygen mode setting
[0042] Three dissolved oxygen change modes were set up, namely, the normal dissolved oxygen C treatment (7.0 mg / L), the rapid hypoxia 24h-rapid reoxygenation 4h AHR treatment, and the slow hypoxia 48h-slow reoxygenation 8h CHR treatment. The normal dissolved oxygen C treatment was used as the control group, and the dissolved oxygen was maintained at 7.0 mg / L; AHR treatment: first, the dissolved oxygen was controlled to drop from 7.0 mg / L to 0.5 mg / L within 4 hours by nitrogen filling, and then the timing was started, and the rapid hypoxia stress was applied for 24 hours, and then aeration was immediately performed to rapidly increase the dissolved oxygen from 0.5 mg / L to 7 mg / L within 4 hours, that is, rapid reoxygenation for 4 hours; CHR treatment: in a water tank with a dissolved oxygen of 7 mg / L, the dissolved oxygen was controlled to slowly drop to 0.5 mg / L within 8 hours by nitrogen filling, and then this hypoxia stress lasted for 48 hours, and then the dissolved oxygen was gradually aerated to slowly increase to 7 mg / L within 8 hours, and the reoxygenation process lasted for 8 hours. Three replicates were set up for each of the three models, each replicate was a 30L plastic tank, each tank was filled with 60 clams. The arrangement of the plastic square tanks was a completely randomized block design. Dissolved oxygen was measured and mortality was counted every 2 hours. No feeding or water changes were done during the treatment period.
[0043] (2) Blood cell collection, fixation and naming
[0044] A small notch was made on the shell edge of the clam near the posterior adductor muscle with a scalpel to facilitate the needle of a 100 μl disposable syringe to penetrate the blood sinus of the posterior adductor muscle to draw hemolymph. Four clams were randomly selected from each treatment group, and the hemolymph was quickly drawn twice and mixed into two 1.5 ml centrifuge tubes with 500 μl of electron microscopy fixative (sterile filtered seawater containing 4% paraformaldehyde, 2.5% glutaraldehyde and 0.05M Tris, pH 7.2) added in advance. The centrifuge tubes were placed on an ice box, and the hemolymph samples were used for scanning of blood cells and transmission electron microscopy preparation, respectively. The samples were finally placed in a 4°C refrigerator and allowed to stand. When the blood cells settled to the bottom of the test tube, the supernatant was carefully drawn and discarded with a pipette, and the fixative was replaced once and stored in a 4°C refrigerator for later use.
[0045] Scanning samples were first post-fixed, i.e. the fixed samples were rinsed 3 times with 0.1M phosphate buffer PBS (PH7.4), 15min each time. 0.1M phosphate buffer PBS (PH7.4) was used to prepare 1% osmium acid and fixed at room temperature in the dark for 1-2h. 0.1M phosphate buffer PBS (PH7.4) was rinsed 3 times, 15min each time. Then gradient dehydration: blood cells were sequentially put into →30% →50% →70% →80% →90% →95% →100% →100% alcohol for dehydration, 15min each time, the above alcohol solution concentration is volume ratio, and then put into pure isoamyl acetate for 15min. Finally, the sample was placed in a critical point dryer (Quorum K850) for drying and ion sputtering (HITACHI MC1000) for gold spraying for about 30s.
[0046] The samples for transmission electron microscopy were first post-fixed, that is, the fixed samples were fixed with 1% osmium acid prepared in 0.1M pH7.4 phosphate buffer PBS at room temperature in the dark for 2 hours. Rinse with 0.1M pH7.4 phosphate buffer PBS 3 times, 15 minutes each time. Then, the samples were dehydrated in a gradient manner at room temperature: blood cells were dehydrated in 30% → 50% → 70% → 80% → 95% → 100% → 100% alcohol in turn, each dehydration was 20 minutes, and dehydrated twice with 100% acetone, each time for 15 minutes. Then, the infiltration embedding was performed, i.e., a solution with a volume ratio of 1:1 of acetone: 812 embedding agent was used, and the embedding agent was treated at 37°C for 2-4h; a solution with a volume ratio of 1:2 of acetone: 812 embedding agent was used, and the embedding agent was infiltrated overnight at 37°C; pure 812 embedding agent was used at 37°C for 5-8h; pure 812 embedding agent was poured into the embedding plate, and the sample was placed in the embedding plate and then placed in an oven at 37°C overnight; the embedding plate was then placed in an oven at 60°C for polymerization for 48h, and the resin block with the sample embedded was taken out for use. The resin block was sliced at 60-80nm on an ultrathin slicer (Leica UC7), and the slice was picked up on a 150-mesh square copper mesh. Slice staining: The copper mesh was stained in a 2% uranyl acetate saturated alcohol solution in the dark for 8min; washed 3 times with 70% alcohol; washed 3 times with ultrapure water; stained 8min with a 2.6% lead citrate solution in the dark for 8min; washed 3 times with ultrapure water, and slightly dried with filter paper. The copper mesh slices were placed in a copper mesh box and dried overnight at room temperature.
[0047] Sample collection time and naming: Samples were collected 24 hours after normal dissolved oxygen C treatment, named CG, and used as the control group; samples were collected 24 hours after rapid hypoxia stress and named AH24h; samples were collected after rapid reoxygenation for 4 hours and named AH24hAR4h; samples were collected after slow hypoxia for 48 hours and named CH48h; samples were collected after slow reoxygenation for 8 hours and named CH48hCR8h.
[0048] (3) Ultrastructural observation and functional classification of blood cells
[0049] 10μl of the fixed SEM sample was slowly added to the blood cell counting plate covered with a cover glass from the sample injection port, and the counting area was found under an ordinary microscope. The various types of cells were counted and statistically analyzed according to the results of SEM observation. The SEM samples were observed under a scanning electron microscope (HITACHI SU8100) and the transmission electron microscope samples were observed under a transmission electron microscope (HITACHI HT7700) and the images were collected. The blood cells were functionally classified according to the changes in cell morphology observed under the SEM and the ultrastructural changes in the granules, mitochondria, endoplasmic reticulum, nucleus and Golgi apparatus in the blood cells observed under the transmission electron microscope.
[0050] result:
[0051] Ⅰ. Classification of blood cell morphology of Philippine clam
[0052] Based on the changes in cell morphology observed under a scanning electron microscope and the characteristics of the intracellular ultrastructure observed under a transmission electron microscope, the hemocytes of the Philippine clam can be divided into five major categories (see Figure 1 , Figure 2 ): (i) amebocytes (AMCs), which often have lamellae or warty pseudopodia on their surface and can move toward foreign bodies or engulf other foreign bodies ( Figure 1 -a, f, k, p, u; Figure 2 -a, f), when the dissolved oxygen changes, the pseudopodia of cells become morphologically strange and their mobility becomes weaker;
[0053] (ii) Secretory cells (SEC) always maintain a spherical shape, and the cell surface generally has many small openings for secretory vesicles to exocytose or secretions attached to them ( Figure 1 -b,f,g,l,q,v; Figure 2 -g), when dissolved oxygen changes, the amount and state of cell surface secretions will change;
[0054] (iii) Thrombocytes (THC), in the normal oxygen group, are spindle-shaped with one end thick and the other end thin, with large nuclei and a long rugby ball shape ( Figure 1 -c), when dissolved oxygen changes, thrombus cells may undergo morphological changes ( Figure 1 -h, m, r, w), affecting blood coagulation function;
[0055] (iv) Spherical cells (SPCs) are normally spherical in shape with a relatively flat cell surface. However, when the dissolved oxygen changes, the cell surface may collapse or bulge, and the spherical cells become irregular ( Figure 1 -d,i,n,s,x);
[0056] (v) Macrophage-like cells (MACs): The cells are generally spherical, with wrinkled cell membranes. The wrinkles are large but regular and symmetrical. When the dissolved oxygen changes, the wrinkles may become messy and irregular due to changes in surface tension, and many depressions may appear on the cell surface. The cells may even lose their spherical structure ( Figure 1 -e, j, o, t, y).
[0057] The most abundant blood cells are spherical cells, accounting for more than one-third, followed by amebocytes, accounting for about one-third, secretory cells and macrophage-like cells, accounting for about one-eighth each, and thrombus cells, accounting for a small proportion, accounting for about one-sixteenth. In addition, there are a small number of spherical small cells ( Figure 1-s), the specific function is unknown. The diameter of various blood cells is small, about 2-3μm, except for the spherical cells, which are about 10μm long. The diameters of secretory cells and spherical cells are relatively uniform, 5-7μm, and the diameters of deformed cells and macrophage-like cells are relatively large, 6-8μm.
[0058] II. Classification of Ultrastructural Characteristics of Blood Cells
[0059] (i) Amoebocytes Figure 2 -a, f) The nucleus is round and generally centered. There are not many particles in the cytoplasm and their sizes and shapes are irregular, with a diameter of 0.2-0.4μm, and they are unevenly distributed in the cell; the mitochondria are also unevenly distributed, often aggregated, and mostly oblong; the endoplasmic reticulum near the nucleus is abundant, the smooth endoplasmic reticulum is mostly round vesicles, and the rough endoplasmic reticulum is mostly slender tubular vesicles; the boundaries between phagocytic vacuoles and pinocytic vacuoles are clear, the phagocytic vacuoles contain granular inclusions, and the pinocytic vacuoles are empty or contain solution, and the cells can be seen engulfing foreign bodies ( Figure 2 -a). Golgi bodies can be seen. The uneven distribution of cell contents may be related to cell deformation, movement and phagocytosis. Therefore, the main function of deformed cells is to phagocytose and remove foreign matter, and the particles in the cells may participate in phagocytosis.
[0060] Rapid hypoxia within 24 hours has little effect on the intracellular ultrastructure, but after rapid reoxygenation, the cells lose the ability to deform and move, and the pseudopodia become weaker in wrapping and engulfing foreign matter due to poor cell membrane fluidity; the outer membrane of intracytoplasmic particles falls off and relaxes; the outer membrane of mitochondria is damaged, or the double membrane cannot be observed, some ridges fall off or are arranged irregularly, and the phagocytic vesicles rupture; the number of endoplasmic reticulum vesicles decreases, the morphology of undamaged endoplasmic reticulum vesicles changes, gaps appear in the cytoplasm, and cells undergo apoptosis.
[0061] (ii) Secretory cells Figure 2 -b, g) has a large, spherical nucleus that is off-center. The granules in the cytoplasm are large and numerous, often accompanied by secretory vesicles, evenly distributed in the cytoplasm, with a diameter of 0.45-0.55μm. There are 13-16 granules in a cell section, which are spherical or crescent-shaped and evenly distributed in the cytoplasm; the mitochondria are oval, and the size distribution is relatively uniform; the secretory vesicles are evenly distributed in the cells, containing abundant small granular secretions. Secretions are common on the cell surface. The endoplasmic reticulum is relatively abundant, with rough and smooth endoplasmic reticulum interwoven, the smooth endoplasmic reticulum is mainly vesicular, and the rough endoplasmic reticulum is mainly tubular. The main function of secretory cells is secretion, and the function of their secretory substances is still unclear. The granules in the cells may be involved in secretory activities.
[0062] Under rapid hypoxia stress, the mitochondrial double membrane is difficult to distinguish, and the cristae of most mitochondria fall off; the endoplasmic reticulum membrane vesicles are unevenly distributed in the cytoplasm, more than 90% of the endoplasmic reticulum is rough endoplasmic reticulum, the outer membrane of the granules falls off, the morphology becomes irregular, and some of the secretory vesicles surrounded by membrane vesicles rupture or fuse with each other, causing cell apoptosis; after slow reoxygenation, the hypoxic damage of the cells is more obvious, the mitochondrial double membrane is not clear, the cristae also fall off, the granules are irregular in shape, and the secretory vesicles surrounded by membrane vesicles have few contents and are unevenly distributed.
[0063] (iii) Thrombocytes Figure 2 -c, h) have large and long nuclei, and the cytoplasm is extremely rich in endoplasmic reticulum and Golgi membrane vesicles. More than 90% of the endoplasmic reticulum is tubular rough endoplasmic reticulum; mitochondria often gather together. There are many unique double-membrane vesicles in thrombus cells, which are either empty or contain solution. There are no particles in the cytoplasm. The main function of thrombus cells is coagulation, and the abundant membrane vesicles lay the foundation for coagulation. Under rapid hypoxia stress, most of the mitochondrial cristae in thrombus cells fall off, the double membrane structure is incomplete, the mitochondria are irregular in shape, and the endoplasmic reticulum membrane vesicles do not undergo obvious changes.
[0064] (iv) Spherical cells ( Figure 2 -d, i): The nucleus is spherical and deviates from the center. Mitochondria are very abundant in the cytoplasm, more than 90% are long strips and easy to aggregate; rare, there are 0-3 small clusters between the nucleus and the cell membrane in the cell section, most of which are slender tubular, and more than 90% of the endoplasmic reticulum are smooth; Golgi bodies are also found in spherical cells; abundant particles, most of which are regular spherical particles in the cytoplasm, with a diameter of 0.35-0.45μm. Under normal dissolved oxygen conditions, there are particles in the cell section, but the number and shape of the particles change with the change of dissolved oxygen; under the change of dissolved oxygen, the damage caused by hypoxia is prominent, the structure of the mitochondrial double membrane and cristae are incomplete, the boundaries of the particles are not clear, the shape is also elongated into an oblong shape, and even diffuse disintegration occurs, the number increases to 9-11, but the shape is different, the number of endoplasmic reticulum membrane vesicles is even rarer, and they are sparsely aggregated into 1-3 piles. It is speculated that the main function of spherical cells is related to the transportation of nutrients and waste.
[0065] (v) Macrophage-like cells ( Figure 2-e, j) have large nuclei with irregular shapes or lobes, and a high nuclear-cytoplasmic ratio. The cell membrane is wrinkled to enclose a number of vacuoles. The rough endoplasmic reticulum in the cell is abundant and almost fills the entire cytoplasm. Ribosome particles are attached to the elongated tubules, and there are also a large number of free ribosome particles in the cytoplasm. The mitochondria are long ovals of uniform size and evenly distributed in the cells, with no particles in the cytoplasm. Regardless of normoxia, hypoxia or reoxygenation, phagocytic vacuoles or pinocytic vacuoles are common in macrophage-like cells. The biggest difference between macrophage-like cells and amebocytes is the presence or absence of particles, and the rough endoplasmic reticulum is the main component in macrophage-like cells. The function of macrophage-like cells is unclear. We speculate that they are mature and aging amebocytes or macrophages.
[0066] The method we invented for functional classification of blood cells based on the ultrastructural changes in blood cells under different patterns of dissolved oxygen changes in marine bivalve molluscs is basically applicable to the analysis and observation of blood cells of all marine bivalve molluscs. It can provide a methodological reference for the protection, identification and immunoanalysis of such species, and is also expected to develop into a routine blood test method for bivalve molluscs.
Claims
1. A method for classifying blood cell functions of Ruditapes philippinarum based on ultrastructural changes, characterized in that: The method comprises the following steps: setting three dissolved oxygen change modes for the cultured Manila clams, which are: maintaining a normal dissolved oxygen of 7.0 mg / L; rapid hypoxia 24h-rapid reoxygenation 4h treatment: firstly controlling the dissolved oxygen to decrease from 7.0 mg / L to 0.5 mg / L within 4h by nitrogen filling method, starting the timing, hypoxia stress for 24h, and then immediately aerating to increase the dissolved oxygen from 0.5 mg / L to 7 mg / L within 4h, i.e. rapid reoxygenation for 4h; slow hypoxia 48h-slow reoxygenation 8h treatment: firstly controlling the dissolved oxygen to decrease slowly to 0.5 mg / L within 8h by nitrogen filling method, then the hypoxia stress is continued for 48h, and then gradually aerating to increase the dissolved oxygen to 7 mg / L within 8h, and the reoxygenation process is continued for 8h; normal dissolved oxygen treatment is used as a control group, and 3 repetitions are set for each of the three modes, and the same number of Manila clams are evenly put into each repetition; dissolved oxygen measurement and death statistics are carried out every 2h; no feeding or water change is performed during the treatment period; Blood was randomly collected from 3-6 Philippine clams in each treatment group, and blood was quickly drawn twice from the blood sinus of the posterior adductor muscle of each Philippine clam. Blood was drawn from the normal dissolved oxygen group 24 hours after treatment, serving as the control group. Blood was drawn from the rapid hypoxia 24 hours-rapid reoxygenation 4 hours treatment group at two time points: 24 hours after rapid hypoxia stress and 4 hours after rapid reoxygenation. Blood was drawn from the slow hypoxia 48 hours-slow reoxygenation 8 hours treatment group at two time points: 48 hours after slow hypoxia and 8 hours after slow reoxygenation. The twice-collected blood from each treatment group was directly and quickly mixed into a container pre-filled with electron microscope fixative, which was placed on ice and finally placed at 4°C for standing. After blood cells were deposited at the bottom of the test tube, the supernatant was drawn and discarded, the fixative was replaced once, and the blood was stored at 4°C for later use. Then, the fixed samples were subjected to scanning electron microscopy and transmission electron microscopy sample preparation, respectively, and then observed under scanning electron microscopy and transmission electron microscopy, respectively. The blood cells were functionally classified according to the changes in cell morphology observed under scanning electron microscopy and the ultrastructural changes in granules, mitochondria, endoplasmic reticulum, cell nucleus and Golgi apparatus in the blood cells observed under transmission electron microscopy. The specific classification standards are morphological standards and ultrastructural standards. The morphological standards are as follows: (1) the cell surface extends with sheet-like or warty pseudopodia, which can move toward foreign bodies or wrap and engulf foreign bodies. When the dissolved oxygen changes, the cell pseudopodia have strange shapes and the mobility becomes weak. Such cells are deformed cells. (2) They always maintain a spherical shape, with small openings for exocytosis of secretory vesicles or secretions adhering to the cell surface. When the dissolved oxygen changes, the amount and state of secretions on the cell surface will change. These cells are secretory cells. (3) In the normal dissolved oxygen group, the cells are spindle-shaped with one end thick and the other end thin, and the nucleus is large and rugby-shaped. When the dissolved oxygen changes, the thrombocytes will undergo morphological changes, affecting the coagulation function. These cells are thrombocytes; (4) The normal morphology is spherical, and the cell surface is relatively flat. However, when the dissolved oxygen changes, the cell surface will collapse or bulge, and the spherical cells will become irregular. These cells are spherical cells. (5) The cells are generally spherical, and the cell membrane is wrinkled. The wrinkles are large but regular and symmetrical. When the dissolved oxygen changes, the wrinkles become messy and irregular due to changes in surface tension, and many depressions appear on the cell surface, or the cells lose their spherical structure. There are pinocytic vacuoles or phagocytic vacuoles in the cells. These cells are macrophage-like cells. The ultrastructural criteria are: (1) The nucleus is round and centered. The granules in the cytoplasm are irregular in size and shape, with a diameter of 0.2-0.4 μm, and are scattered in the cytoplasm. The mitochondria are unevenly distributed or clustered together, mostly in an oblong shape. The endoplasmic reticulum near the nucleus is abundant, the smooth endoplasmic reticulum is round and vesicular, and the rough endoplasmic reticulum is elongated and tubular. The boundaries between phagocytic vacuoles and pinocytic vacuoles are clear, the phagocytic vacuoles contain granular inclusions, and the pinocytic vacuoles are empty or contain solution. The cells can be seen engulfing foreign bodies, and there are Golgi bodies in the cytoplasm. Within 24 hours Rapid hypoxia has little effect on the ultrastructure of cells, but after rapid reoxygenation, cells lose the ability to deform and move. The pseudopodia have poor membrane fluidity and their ability to engulf foreign matter becomes weaker. The outer membrane of particles in the cytoplasm falls off and becomes loose. The outer membrane of mitochondria is damaged, or the double membrane cannot be observed. Some cristae fall off or are arranged irregularly, and phagocytic vacuoles rupture. The number of endoplasmic reticulum vesicles decreases, and the morphology of undamaged endoplasmic reticulum vesicles changes. There are gaps in the cytoplasm, and cells undergo apoptosis. These cells are deformed cells. (2) The nucleus is large, spherical, and off-center; the granules in the cytoplasm are large and numerous, evenly distributed in the cytoplasm, with a diameter of 0.45-0.55 μm. There are 13-16 granules in a cell section, which are spherical or half-moon shaped, accompanied by secretory vesicles; the mitochondria are oval, with uniform size distribution; the secretory vesicles are also evenly distributed in the cell, containing abundant fine granular secretions, and secretions are seen on the cell surface; the endoplasmic reticulum is abundant, with rough and smooth endoplasmic reticulum interwoven, the smooth endoplasmic reticulum is vesicular, and the rough endoplasmic reticulum is tubular; under rapid hypoxia stress, the mitochondrial double membrane is difficult to distinguish, and the cristae of most mitochondria fall off; the endoplasmic reticulum membrane vesicles are unevenly distributed in the cytoplasm, with more than 90% of the endoplasmic reticulum being rough endoplasmic reticulum, the outer membrane of the granules falls off, the morphology becomes irregular, and some secretory vesicles surrounded by membrane vesicles rupture or fuse with each other, causing cell apoptosis; After slow reoxygenation, the hypoxic damage of cells is more obvious, the mitochondrial double membrane is unclear, the cristae are also detached, the granules are irregular in shape, and the contents of the secretory vesicles surrounded by membrane vesicles are few and unevenly arranged. These cells are secretory cells; (3) The nucleus is large and long, and the cytoplasm is rich in endoplasmic reticulum and Golgi membrane vesicles. More than 90% of the endoplasmic reticulum is tubular rough endoplasmic reticulum. The mitochondria are clustered together, and there are unique double-membrane vesicles in the cells. The vesicles are either empty or contain solution, and there are no particles in the cytoplasm. These cells are thrombocytes. Under rapid hypoxia stress, the cristae of most mitochondria in thrombocytes fall off, the double membrane structure is incomplete, the mitochondria are irregular in shape, and the endoplasmic reticulum membrane vesicles do not undergo obvious changes. (4) The cell nucleus is spherical and deviates from the center. The mitochondria in the cytoplasm are very abundant, more than 90% of which are long strips and easy to aggregate. The endoplasmic reticulum is scarce. There are 0-3 small clusters between the nucleus and the cell membrane in the cell section, most of which are slender tubular. More than 90% of the endoplasmic reticulum is smooth. There is a Golgi body in the cytoplasm. The particles in the cytoplasm are mostly regular spherical, with a diameter of 0.35-0.45μm. Under normal dissolved oxygen conditions, there are 3-5 spherical particles in the cell section, but the number and shape of the particles change with changes in dissolved oxygen. Under changes in dissolved oxygen, the damage caused by hypoxia is prominent, the structure of the mitochondrial double membrane and cristae are incomplete, the boundaries of the particles are not clear, the shape is elongated into an oblong shape, and even diffuse disintegration occurs. The number increases to 9-11, but the shapes are different. The number of endoplasmic reticulum membrane vesicles is even scarcer, and they are sparsely aggregated into 1-3 piles. This type of cell is a spherical cell. (5) The nucleus is large, irregular in shape, or lobed, with a high nuclear-cytoplasm ratio; the cell membrane is wrinkled to form vacuoles, the rough endoplasmic reticulum is abundant in the cell, almost filling the entire cytoplasm, ribosome particles are attached to the elongated tubules, and there are also a large number of free ribosome particles in the cytoplasm; the mitochondria are long ovals of uniform size and evenly distributed in the cell; there are no particles in the cytoplasm; regardless of normoxia, hypoxia or reoxygenation, phagocytic vacuoles or pinocytic vacuoles are found in the cells, and these cells are macrophages.
2. The method for classifying blood cell functions of Ruditapes philippinarum based on ultrastructural changes according to claim 1, characterized in that: The Manila clams were not fed 24 h before the experiment.
3. The method for classifying blood cell functions of Ruditapes philippinarum based on ultrastructural changes according to claim 1, characterized in that: The electron microscope fixing solution contains 4% paraformaldehyde by volume, 2.5% glutaraldehyde by volume and sterile filtered seawater with a molar concentration of 0.05M Tris, with a pH of 7.
2.
4. The method for classifying blood cell functions of Ruditapes philippinarum based on ultrastructural changes according to claim 1, characterized in that: Scanning electron microscope sample preparation and observation: rinse the fixed sample 3 times with 0.1M phosphate buffer (PBS) at pH 7.4, each time for 15 min, then add it to 0.1M phosphate buffer (PBS) containing 1% osmium acid by mass and fix it at room temperature in the dark for 1-2 h, rinse it again with 0.1M phosphate buffer (PBS) at pH 7.4 for 3 times, each time for 15 min; then dehydrate it in a gradient manner: blood cells are placed in →30% →50% →70% →80% →90% →95% →100% →100% alcohol in sequence, each time for 15 min; then put it in pure isoamyl acetate for 15 min; finally, put the sample in a critical point dryer for drying and spray gold on an ion sputtering instrument for 30 s, and then observe and collect images under a scanning electron microscope.
5. The method for classifying blood cell functions of Ruditapes philippinarum based on ultrastructural changes according to claim 1, characterized in that: Transmission electron microscopy sample preparation and observation: The fixed samples were fixed with 1% osmium acid prepared in 0.1M pH 7.4 phosphate buffer PBS in the dark for 2 hours at room temperature, and then rinsed with 0.1M pH 7.4 phosphate buffer PBS for 3 times, 15 minutes each time; then dehydrated in a gradient manner at room temperature: blood cells were dehydrated in 30% → 50% → 70% → 80% → 95% → 100% → 100% alcohol in turn, each dehydration for 20 minutes, and dehydrated twice with 100% acetone, each for 15 minutes; then infiltration embedding, that is, the volume ratio of acetone: 812 embedding agent was 1:1, and the reaction was carried out at 37°C for 2-4 hours; the volume ratio of acetone: 812 embedding agent was 1:2, and infiltration was carried out overnight at 37°C; pure 812 embedding agent Embed at 37℃ for 5-8h; then pour pure 812 embedding agent into the embedding plate, put the sample into the embedding plate and keep it at 37℃ overnight; then put the embedding plate at 60℃ for polymerization for 48h, take out the resin block with the sample for use; then ultrathin sectioning: the resin block is sliced at 60-80nm on an ultrathin slicer, and the slice is caught on a 150-mesh square-hua membrane copper mesh; section staining: the copper mesh is stained with a 2% by volume uranyl acetate saturated alcohol solution in the dark for 8min; washed 3 times with 70% by volume alcohol; washed 3 times with ultrapure water; stained with a 2.6% by mass lead citrate solution in the dark for 8min; washed 3 times with ultrapure water, and slightly dried with filter paper; the copper mesh sections are placed in a copper mesh box and dried overnight at room temperature; finally, observed under a transmission electron microscope, and image analysis was collected.