Non-destructive marking method for razor clam species

Abstract

The application discloses a non-destructive marking method for razor clam, which can remove the fluff on the surface of the razor clam without affecting the growth and survival of the razor clam. Even without using chemical reagents, the method can remove the left shell fluff, the right shell fluff or the fluff on both the left and right shells to realize non-destructive marking of the razor clam, and the marking can last for more than 5 months. The method has the advantages of obvious marking, low cost, simple operation, avoidance of physiological damage to the razor clam and long-lasting retention. In particular, the method uses a file to gently polish the shell along the radial rib at an angle of 30-45 degrees in seawater, and the degree of shell damage is small and the efficiency of fluff removal is high. Since the method can effectively remove the fluff on the surface of the razor clam, it can not only realize non-destructive marking by removing the fluff, but also further mark the razor clam with removed surface fluff by using a chemical method, thereby effectively increasing the types of marking.

Description

Technical Field

[0001] This invention relates to a method for marking shellfish, and more particularly to a non-destructive marking method for shellfish of the genus *Ceratophyllum*. Background Technology

[0002] In ecology, molecular biology, and aquaculture breeding, to ensure consistent living conditions, it is necessary to raise different families and treatment groups of the same species in the same environment. To facilitate differentiation between different families and treatment groups, it is necessary to label the shellfish. Existing simple shellfish labeling methods include tagging, separate labeling, and chemical labeling.

[0003] Tagging is a physical method of marking animals externally. A common method involves using a punch to create a hole in the shell of a mollusk between the edge and the hinge. A tag is then attached to the hole using thread, wire, or barbs. Tags are typically made of plastic, but silver or other metals are also used. They often have different codes or patterns and come in various shapes, such as circles, rectangles, ovals, and noodle shapes. Tagging is quick, intuitive, and long-lasting, making it suitable for long-term research. However, only larger individuals can withstand the manipulation pressure and additional metabolic burden of tag attachment. Even then, tagging can cause physiological damage to mollusks, affecting their growth and development. Therefore, tagging is rarely used in pond aquaculture breeding.

[0004] Molecular markers are genetic markers based on nucleotide sequence variations within the genetic material of individuals, directly reflecting genetic polymorphism at the DNA level. Molecular marker development requires tissue sampling of the mollusks to be tagged (common tissue sites include gills, adductor muscle, gonads, and mantle). By designing different primers, relevant bands are amplified on the organism's genomic DNA using PCR. Differences in nucleotide sequences are then identified and analyzed using electrophoresis and software, and can be used as markers for the study of biological genetic information. While this method has a wide range of applications and significant effects, it not only causes physiological damage to mollusks, similar to tagging, but is also time-consuming and costly, and suffers from difficulties in marker selection.

[0005] Chemical marking involves cleaning the shell surface and then soaking it in calcein or coating it with a non-toxic, transparent epoxy resin solution to form a mark. This method has advantages such as clear marking, low cost, simple operation, avoidance of physiological damage to shellfish, and long-lasting effect.

[0006] genus *Ceratophyllum* ( ScapharcaMussels (including blood clams and cockles) are widely distributed and abundant. They are not only delicious but also highly nutritious, containing high protein, low fat, and high vitamins, making them important economic shellfish. One distinguishing feature from other shellfish is the black or grayish-brown velvet covering their shells. This is particularly true for blood clams with shells longer than 25 mm, where multiple radial ribs are more prominent, resulting in varying shades of velvet that are difficult to remove. To date, no method for removing this velvet without affecting the growth and survival of the shellfish has been found. This makes it impossible to use non-destructive chemical labeling methods for blood clams, presenting problems associated with tagging and molecular labeling. Summary of the Invention

[0007] The present invention aims to solve the above-mentioned technical problems existing in the prior art by providing a non-destructive marking method for mollusks of the genus *Ceratophyllum*.

[0008] The technical solution of this invention is: a non-destructive marking method for mollusks of the genus *Vibrio vulgaris*, which is carried out in the following steps:

[0009] Step 1. Cleaning shellfish

[0010] Select blood clam species with a shell length of at least 25 mm, wash them with seawater to remove dirt and attached substances from the shell surface;

[0011] Step 2. Remove the fuzz from the surface of the shell.

[0012] Place the shellfish in seawater, hold a file at a 30-45° angle to the shell surface, and polish along the radial ribs of the shell to remove the hairs on the shell surface until no obvious hairs remain on the shell surface when observed with the naked eye.

[0013] Step 3. Microscopic examination

[0014] Observe the shells of the shellfish under a microscope, select shellfish with less than 5% shell damage, and use the tip of a scalpel to remove the remaining hairs until less than 10% of the hairs remain on the shell surface under the microscope;

[0015] Step 4. Temporary Rest and Recovery

[0016] The shellfish were temporarily kept for 3 days, with the water changed once a day during the period, and half of the water was replaced. Algae powder was fed to them daily. After 3 days, the shellfish that showed normal feeding and burrowing behavior were selected and marked.

[0017] The preferred technical solution is to temporarily raise the shellfish after step 1 until the mortality rate stabilizes. During the temporary raising period, the water is changed once a day, with half of the water being changed. Algae powder is fed daily at a rate of 1% of the shellfish's weight. Dead shellfish are removed after the temporary raising period.

[0018] The preferred technical solution is that the algae powder being fed is a mixture of Spirulina, Chlorella, and Dunaliella salina.

[0019] This invention removes the fuzz from the surface of blood clams without affecting their growth and survival. Even without chemical reagents, it can achieve non-destructive marking of blood clams by removing fuzz from the left or right shell, or both shells simultaneously, with the marking lasting at least 5 months. It offers advantages such as clear marking, low cost, simple operation, avoidance of physiological damage to the clams, and long-lasting results. In particular, using a file at a 30-45° angle in seawater to gently polish the shell along its radial ribs minimizes shell damage and maximizes fuzz removal efficiency. Because this invention effectively removes fuzz from the surface of blood clams, in addition to non-destructive marking through fuzz removal, the fuzz-removed blood clams can be further marked using chemical methods, effectively increasing the variety of marking options. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the operation of removing the fuzz from the surface of the cockle shell using a file, as described in Embodiment 1 of the present invention.

[0021] Figure 2 This is a schematic diagram illustrating the process of peeling the villi from the shell of a cockle under a stereomicroscope in Embodiment 1 of the present invention.

[0022] Figure 3 This is a diagram illustrating the marking effect of the cockle in Embodiment 1 of the present invention.

[0023] Figure 4 These are the effect diagrams of removing the surface hairs of cockle shells in Embodiment 1 and the comparative example of the present invention.

[0024] Figure 5 This is a diagram illustrating the marking effect of blood clam in Embodiment 2 of the present invention.

[0025] Figure 6 This is a comparison diagram of the regeneration of the left shell villi of the cockle in Embodiment 1 of the present invention after 5 months. Detailed Implementation Example 1

[0026] The present invention provides a non-destructive marking method for mollusks of the genus *Vibrio vulgaris*, which is performed in the following steps:

[0027] Step 1. Cleaning shellfish

[0028] Select cockles with a shell length of 42.00 ± 1.27 mm, wash them with seawater to remove dirt and attached substances from the shell surface; to reduce the mortality rate after marking, it is best to temporarily hold the shellfish until the mortality rate stabilizes, generally for about 3 days. During the temporary holding period, change the water once a day, replacing 1 / 2 of the water volume, and feed them algae powder daily. The algae powder is preferably a mixture of spirulina, chlorella, and Dunaliella salina, and the feeding amount is 1% of the shellfish's weight. After the temporary holding period, remove any dead cockles.

[0029] Step 2. Remove the fuzz from the surface of the shell.

[0030] Take the cockles and place them in seawater, as... Figure 1 As shown, a 5×180 mm pointed flat file was used to gently polish the surface of the left shell along the radial ribs of the shell at a 30-45° angle for 5 minutes. No obvious hairs remained on the surface of the left shell when observed with the naked eye. Removing the hairs on the shell surface in seawater can prevent dew stress caused by marking.

[0031] Step 3. Microscopic examination

[0032] according to Figure 2 As shown, the shellfish were placed under a stereomicroscope to observe the shells. Shellfish with less than 5% shell damage were selected, and the remaining hairs were gently picked off with the tip of a scalpel until less than 10% of the hairs remained on the shell surface under the microscope.

[0033] Step 4. Temporary Rest and Recovery

[0034] The shellfish were temporarily held for 3 days, with half of the water changed daily during this period. They were fed a mixture of spirulina, chlorella, and Dunaliella salina powder daily. After 3 days, the shellfish's feeding and burrowing behaviors returned to normal, at which point they were tagged. The tagging effect was as follows: Figure 3 As shown.

[0035] Comparative example:

[0036] Take two cockles from Example 1 that have been temporarily kept in step 1, place them in seawater, hold a 1.4×13.6 cm electric razor with the blade parallel to the radial ribs of the cockle shell, and remove the hairs from the left shell along the radial ribs for 5 minutes. Then, place the shells under a stereomicroscope to observe them, following the method in step 3.

[0037] The processed cockles of Embodiment 1 and the comparative example are shown below. Figure 4 As shown, Figure 4 In Example 1, A is the pointed flat file used in this invention; B is the cockle in Example 1 of this invention without the fuzz removed; C is the cockle in Example 1 of this invention after the fuzz has been removed; D is the electric razor used in the comparative example; E is the cockle in the comparative example without the fuzz removed; and F is the cockle in the comparative example after the fuzz has been removed. The results show that there is a significant difference in the amount of fuzz remaining after 5 minutes of fuzz removal between Example 1 and the comparative example. In Example 1, no obvious fuzz residue was observed on the shell surface with the naked eye, and the residue was less than 18% under a microscope, while in the comparative example, 70% remained. Example 2

[0038] Step 1. Cleaning shellfish

[0039] Select blood clams with a shell length of 29.2 mm, wash them with seawater to remove dirt and attached substances from the shell surface; in order to reduce the mortality rate after marking, it is best to temporarily hold the shellfish until the mortality rate stabilizes, generally for about 3 days. During the temporary holding period, change the water once a day, replacing 1 / 2 of the water volume, and feed them algae powder daily. The algae powder is preferably a mixture of spirulina, chlorella, and Dunaliella salina, and the feeding amount is 1% of the shellfish's weight. After the temporary holding period, remove the dead blood clams.

[0040] Step 2. Remove the fuzz from the surface of the shell.

[0041] Place the blood clam in seawater, and gently polish the surface of the left and right shells at a 30-45° angle along the radial ribs of the shell with a 3×140 mm pointed flat file. Visually inspect the surface of the left shell and find that there are no obvious hairs left. Removing the hairs from the shell surface in seawater can prevent the dew stress caused by marking.

[0042] Step 3. Microscopic examination

[0043] Observe the shells of the shellfish under a stereomicroscope, select shellfish with less than 5% shell damage, and gently remove less than 13% of the remaining hairs with the tip of a scalpel until less than 10% of the hairs remain on the shell surface under the microscope;

[0044] Step 4. Temporary Rest and Recovery

[0045] The shellfish were temporarily held for 3 days, with half of the water changed daily during this period. They were fed a mixture of spirulina, chlorella, and Dunaliella salina powder daily. After 3 days, the shellfish's feeding and burrowing behaviors returned to normal, at which point they were tagged. The tagging effect was as follows: Figure 5 As shown. Example 3

[0046] Step 1. Cleaning shellfish

[0047] Select cockles with a shell length of 38.88 mm, wash them with seawater to remove dirt and attached substances from the shell surface; in order to reduce the mortality rate after marking, it is best to temporarily hold the shellfish until the mortality rate stabilizes, generally for about 3 days. During the temporary holding period, change the water once a day, replacing 1 / 2 of the water volume, and feed them algae powder daily. The algae powder is preferably a mixture of spirulina, chlorella, and Dunaliella salina, and the feeding amount is 1% of the shellfish's weight. After the temporary holding period, remove any dead cockles.

[0048] Step 2. Remove the fuzz from the surface of the shell.

[0049] Place the blood clam in seawater, and gently polish the surface of the right shell along the radial ribs of the shell at a 30-45° angle with a 4×160 mm pointed flat file. Visually observe that there are no obvious hairs remaining on the surface of the left shell. Removing the hairs on the shell surface in seawater can prevent the dew stress caused by marking.

[0050] Step 3. Microscopic examination

[0051] Observe the shells of the shellfish under a stereomicroscope, select shellfish with less than 5% shell damage, and gently pick away less than 16% of the remaining hairs with the tip of a scalpel until less than 10% of the hairs remain on the shell surface under the microscope. Since the remaining shell hairs after polishing are generally shorter, the peeling efficiency is higher and the damage to the shell is less.

[0052] Step 4. Temporary Rest and Recovery

[0053] The shellfish were temporarily kept for 3 days, with half of the water changed daily during this period. They were also fed algae powder daily. After 3 days, the shellfish's feeding and burrowing behaviors were normal, and they were then tagged.

[0054] experiment:

[0055] Clams with a shell length of 40.51 ± 3.63 mm were selected. Three experimental groups were set up: one group with the left shell hairs removed, one group with the right shell hairs removed, and one group with all hairs removed. There was also one control group with no hairs removed. Each treatment group had three replicates, and each group had 10 clams.

[0056] The three experimental groups—left shell velvet removal, right shell velvet removal, and all velvet removal—were conducted according to the following methods:

[0057] Step 1. Wash with seawater to remove dirt and attached substances from the surface of the shells; temporarily hold the shellfish for 3 days, changing the water once a day during the holding period, replacing 1 / 2 of the water volume, and feeding them algae powder daily. The algae powder is a mixture of spirulina, chlorella, and Dunaliella salina, and the feeding amount is 1% of the shellfish's weight. After the holding period, remove any dead shellfish.

[0058] Step 2. Remove the fuzz from the surface of the shell.

[0059] Take the cockles and place them in seawater. Hold a 4×160 mm pointed flat file and gently polish the surface of the left, right or both shells at a 30-45° angle along the radial ribs of the shell. Visually inspect the shell surface and find that there are no obvious hairs left. Removing the hairs from the shell surface in seawater can prevent the dew stress caused by marking.

[0060] Step 3. Microscopic examination

[0061] Observe the shells of the shellfish under a stereomicroscope, select shellfish with less than 5% shell damage, and gently remove the remaining hairs with the tip of a scalpel until less than 10% of the hairs remain on the shell surface under the microscope;

[0062] Step 4. Temporary Rest and Recovery

[0063] The shellfish were temporarily kept for 3 days, with half of the water changed daily during this period. They were also fed algae powder daily. After 3 days, the shellfish's feeding and burrowing behaviors were normal, and they were then tagged.

[0064] The control group was washed with seawater to remove dirt and attached substances from the shells. The shellfish were temporarily held, with the water changed once a day, and half of the water was replaced. They were fed algae powder daily, which was a mixture of spirulina, chlorella, and Dunaliella salina. The amount of algae powder fed was 1% of the shellfish's weight. The temporary holding period ended when the experimental group was marked.

[0065] Five months after marking, there was no significant difference in survival rate between the groups and the control group (see Table 1); there were also no significant differences in growth traits such as shell length and shell height between the groups and the control group (see Table 2).

[0066] Table 1. Survival rate (%) of cockles 5 months after tagging

[0067]

[0068] Comparing the regeneration of villi on the surface of cockles immediately after removal from the shells in Example 1 and after 5 months of temporary rearing, as shown... Figure 6 As shown. Figure 6 In the cases A, B, and C, the velvet on the left shell of the cockle had just been removed; in the cases D, E, and F, the velvet on the left shell of the cockle had been removed for 5 months. The results showed that there was no obvious regeneration of the velvet on the surface of the cockle shell after 5 months of marking.

[0069] In summary, this invention has no significant impact on the growth and survival of blood clam species, and the marking can last for at least 5 months.

Claims

1. A non-destructive marking method for mollusks of the genus *Ceratophyllum*, characterized in that... Follow these steps in sequence: Step 1. Cleaning shellfish Select blood clam species with a shell length of at least 25 mm, wash them with seawater to remove dirt and attached substances from the shell surface; Step 2. Remove the fuzz from the surface of the shell. Place the shellfish in seawater, hold a file at a 30-45° angle to the shell surface, and polish along the radial ribs of the shell to remove the fuzz on the shell surface until no obvious fuzz remains on the shell surface can be observed with the naked eye. Step 3. Microscopic examination Observe the shells of the shellfish under a microscope, select shellfish with less than 5% shell damage, and use the tip of a scalpel to remove the remaining hairs until less than 10% of the hairs remain on the shell surface under the microscope; Step 4. Temporary Rest and Recovery The shellfish were temporarily kept for 3 days, with the water changed once a day during the period, and half of the water was replaced. Algae powder was fed to them daily. After 3 days, the shellfish that showed normal feeding and burrowing behavior were selected and marked.

2. The non-destructive marking method for clams according to claim 1, characterized in that: After step 1, the shellfish are temporarily held until the mortality rate stabilizes. During the holding period, the water is changed once a day, with half of the water being replaced. Algae powder is fed daily at a rate of 1% of the shellfish's weight. Dead shellfish are removed after the holding period.

3. The non-destructive marking method for clams according to claim 1 or 2, characterized in that: The algae powder being fed is a mixture of spirulina, chlorella, and Dunaliella salina.

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