An exogenous injection neurotransmitter for promoting mussel byssus secretion and its screening method and application
By injecting adrenaline into the mussel's closure muscle, the problem of insufficient secretion of mussel foot silk was solved, significantly increasing the amount of mussel foot silk secretion and increasing the breeding yield.
Patent Information
- Application Number
- CN202410340422.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-25
- Publication Date
- 2025-04-08
- Estimated Expiration
- 2044-03-25
AI Technical Summary
The prior art has not yet explored the use of neurotransmitters to stimulate the secretion of foot silk of mussel foot tissue, resulting in the limited secretion of foot silk of mussel foot silk and affecting the breeding yield.
Promotes the secretion of its foot filament by injecting specific neurotransmitters, such as epinephrine (EPI) into the mussel's cuff muscle.
After injection of 10-3M adrenaline, the amount of foot silk secretion in mussels was significantly higher than that in the control group, which solved the problem of insufficient foot silk secretion in mussels and increased the breeding yield.
Smart Images
Figure CN118236360B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to an exogenous injection neurotransmitter that promotes mussels to secrete byssus, and a screening method and application thereof. Background Art
[0002] Thick-shelled mussel (Mytilus coruscus) belongs to the phylum Mollusca, class Bivalvia, order Mytiloida, family Mytilidae, genus Mytilus.
[0003] Thick-shelled mussels need to go through an irreversible process of attachment metamorphosis in their life history, starting from fertilized eggs to the planktonic larvae of the eyed larvae, where they undergo attachment metamorphosis and then become juveniles and adults living benthic. Mussels secrete byssal protein through glands in their foot tissues, which solidify in seawater to form tough byssal threads that attach themselves to solid attachment substrates.
[0004] The attachment of mussel byssus is closely related to the yield of mussel farming. Mussels attach to the farming ropes by secreting byssus until fishermen unhook the bunches of mussels from the floating rafts to complete the harvest. If the mussels fall from the farming ropes, it will directly affect the yield of farmed mussels. In the field of marine bioengineering, byssus is a unique biomaterial with excellent adhesion and elastic properties. In recent years, the byssus attachment mechanism has become a research hotspot in the field of biomaterials. Although the secretion of byssus by mussels is an individual active process, it is still unknown which endogenous factor in the body regulates the secretion of mussel byssus.
[0005] At present, the research on the amount of mussel byssus secretion is limited to the environmental field, such as temperature, salinity, etc., but the research based on the use of specific neurotransmitters or their analogues to stimulate the secretion of byssus byssus by mussel foot tissue has not been reported. Summary of the invention
[0006] The present invention aims to provide an exogenous injection neurotransmitter for promoting the secretion of byssus by mussels, and a screening method and application thereof, involving the use of specific neurotransmitters or their analogs to stimulate mussels, thereby increasing the amount of mussel byssus secretion, and achieving significant promotion of thick-shelled clams' secretion of byssus by injecting neurotransmitters.
[0007] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:
[0008] The first aspect of the present invention is to provide an exogenous injection neurotransmitter for promoting the secretion of byssus by mussels, wherein the exogenous injection neurotransmitter is epinephrine (EPI).
[0009] The second aspect of the present invention is to provide a screening method for exogenously injected neurotransmitter EPI, comprising the following steps:
[0010] S1. Screening of mussels: Two-year-old thick-shelled mussels with a shell length of 7-8 cm were selected as experimental subjects;
[0011] S2. Determination of neurotransmitter content in different stages of mussel byssus secretion: The foot tissues of mussels in the resting stage, foot exploration stage, byssus secretion stage, and late byssus secretion stage were used as samples to determine the neurotransmitter content. The results showed that the relative content of adrenaline (EPI) and serotonin (5-HT) in the foot exploration stage was significantly higher than that in the resting stage;
[0012] S3. Determination of changes in the amount of mussel byssus secretion after neurotransmitter injection: The neurotransmitters adrenaline and serotonin were injected into mussels, and the amount of mussel byssus secretion was counted at different time points after injection. The results showed that after 10 -3 72 hours after M-epinephrine treatment, the amount of byssus secreted by mussels was significantly higher than that in the control group, while serotonin injection had no significant effect in promoting byssus secretion.
[0013] Preferably, in step S2, the mussels are placed in water and allowed to rest for 5-10 minutes to obtain foot tissue as a resting period sample; the foot tissue is obtained when the mussels extend their feet outside the body to explore, and is used as a foot exploration period sample; the foot tissue is obtained when the mussels secrete byssus threads, and is used as a byssus thread secretion period sample; and the foot tissue is obtained 5-10 minutes after the mussels have retracted their byssus threads after secreting them, and is used as a late byssus thread secretion period sample.
[0014] Preferably, in step S2, all collected mussel foot tissues need to be quickly placed in liquid nitrogen, and the number of foot tissue samples at different stages needs to be greater than or equal to 6.
[0015] Preferably, in step S3, the injection site of the neurotransmitter is the adductor muscle of the mussel. In terms of the selection of the injection site, the inventors have also experimented with injecting neurotransmitters into the feet, and both excessive and no secretion of the byssus occurred, and the expression was not stable; moreover, the feet are difficult to be clearly observed, and the injection is difficult. The angle of the needle will affect the injection site, and it is easy to inject into other muscle tissues around the foot. Thick-shelled mussels have an open-tube circulatory system, and body fluids and blood circulate together. The adductor muscle is the blood storage site of mussels. Injecting exogenous neurotransmitters here can effectively and quickly transmit neurotransmitters. In addition, the adductor muscle is obvious, and there will be no injection in the wrong position. The secretion is stable, and the adductor muscle is finally selected as the injection site.
[0016] Preferably, in step S3, adrenaline and serotonin powders are dissolved in mussel saline and then injected. The ingredients of the mussel saline include: 20mM HEPES, 436mM NaCL, 53mM MgSO4, 10mM KCL, 10mM CaCl2. This formula fits the ion content of mussel body fluid so as to better control variables.
[0017] Preferably, in step S3, the concentration of adrenaline and serotonin in the injection is 10 -2 M~10 -5 M, injection volume is 200 μL.
[0018] Preferably, in step S3, the number of byssus threads secreted by the mussels is counted at 6h, 12h, 24h, 48h and 72h after injection.
[0019] The third aspect of the present invention is to provide the application of the above-mentioned exogenous injection neurotransmitter for promoting the secretion of byssus by mussels, wherein the application method is to inject adrenaline into the adductor muscle of the mussel to promote the secretion of its byssus byssus.
[0020] The present invention has the following beneficial effects:
[0021] The present invention collected the foot tissues of thick-shelled mussels at different stages of byssus secretion (1. static stage; 2. foot exploration stage; 3. byssus secretion stage; 4. late byssus secretion stage) and conducted targeted neurotransmitter analysis, and found that the relative content of adrenaline and serotonin in the thick-shelled mussel foot exploration stage was significantly increased compared with the static stage (P<0.05). This was verified by injecting adrenaline and serotonin into the adductor muscle of thick-shelled mussels. The results showed that after 10 injections of adrenaline and serotonin, the adductor muscle of thick-shelled mussels was significantly increased. -3 After 72h of MEPI, the amount of byssus secreted by mussels was significantly higher than that in the control group (P<0.05), but 5-HT injection had no significant effect in promoting byssus secretion (P>0.05).
[0022] The present invention is the first to discover that the injection of exogenous neurotransmitter adrenaline can significantly promote the secretion of byssus in thick-shelled mussels, which has not been reported in any shellfish, thus filling the research gap. The method of the present invention involves using specific neurotransmitters or their analogs to stimulate mussels, thereby increasing the amount of mussel byssus secretion, laying an important foundation for studying the secretion mechanism of mussel byssus and its physiological regulation. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is a heat map of total neurotransmitters during different stages of thick-shelled mussel byssus secretion provided by an embodiment of the present invention.
[0024] In the figure, the horizontal axis represents 6 tissue samples at different periods; the vertical axis represents different neurotransmitters; the color represents the expression level of neurotransmitters, red represents high expression, and the darker the red, the higher the expression level, and blue represents low expression.
[0025] Figure 2 This is a graph showing the relative content changes of neurotransmitters during different stages of byssus secretion of thick-shelled mussels provided by an embodiment of the present invention.
[0026] Figure 3This is a graph showing changes in the amount of byssus secretion at different time points after exogenous injection of adrenaline into thick-shelled mussels provided in an embodiment of the present invention.
[0027] Figure 4 This is a graph showing changes in the amount of byssus secretion at different time points after exogenous injection of serotonin in thick-shelled mussels provided by an embodiment of the present invention. Figure 2-4 In the table, significance is marked by lowercase letters abc, and there are significant differences among the treatment groups marked with different letters, P < 0.05. DETAILED DESCRIPTION
[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.
[0029] Example
[0030] This embodiment provides a method for screening exogenously injected neurotransmitters that promote mussels to secrete byssus, comprising the following steps:
[0031] S1. Screening of mussels
[0032] Thick-shelled mussels were collected from Gouqi Island, Shengsi County, Zhejiang Province. The attachments on the shell surface were removed and the excess byssus were cut along the edge of the mussel shell to separate them into individual mussels. The mussels were then placed in a plastic basin containing 10L natural seawater (salinity 30‰) and temporarily cultured for 1 week at 21°C, during which the seawater was changed once a day. They were fed with Qingdao giant flat algae (Isochrysiszhanjiangensis) and ball-shaped flagella (Isochrysis galbana) every day. Two-year-old thick-shelled mussels with a shell length of 7-8cm were selected for neurotransmitter content determination and neurotransmitter injection experiments.
[0033] S2. Determination of neurotransmitter content at different stages of mussel byssus secretion
[0034] (1) After 1 week of temporary culture, mussels that secrete byssus were selected from thick-shelled mussels. The secreted byssus were removed and the mussels were placed in a glass culture dish and placed in a plastic basin containing 10L natural seawater (salinity 30‰), with 7 mussels in each basin, for a total of 6 basins. During the experiment, the seawater was replaced daily, the seawater was pumped, and Qingdao giant flat algae (I.zhanjiangensis) and ball whip golden algae (Isochrysis galbana) were fed three times a day.
[0035] (2) Place the mussels in seawater and let them rest for 5-10 minutes, remove the mussels that have not extended their feet, and collect their foot tissue as the resting period sample; when the mussel foot tissue extends out of the body to explore but does not secrete byssal threads, collect its foot tissue as the foot exploration period sample; when the mussel foot explores in vitro and secretes byssal threads, collect its foot tissue as the byssal thread secretion period sample; when the mussel secretes byssal threads and the foot retracts into the body for 5-10 minutes and does not extend again, collect its foot tissue as the byssal thread late secretion period sample. Six foot tissue samples were collected for each period, and the tissues were quickly frozen in liquid nitrogen. After the tissues were ground, the neurotransmitter content was determined using a liquid chromatograph and a mass spectrometer.
[0036] (3) From the total heat map of neurotransmitters ( Figure 1 ) and box plots ( Figure 2 ) It can be seen that the relative contents of adrenaline and serotonin in the foot exploration period were significantly higher than those in the foot resting period (P<0.05), indicating that the content of adrenaline and serotonin in the thick-shelled mussel foot increased significantly during the in vitro exploration stage. We speculate that adrenaline and serotonin are involved in the secretion of mussel byssus.
[0037] S3. Determination of changes in the amount of mussel byssus secretion after neurotransmitter injection
[0038] (1) To investigate the relationship between the injection of neurotransmitters and the secretion of byssus in thick-shelled mussels, adrenaline and serotonin powder were dissolved in mussel saline (20 mM HEPES, 436 mM NaCL, 53 mM MgSO4, 10 mM KCL, 10 mM CaCl2) at concentrations of 10 -2 M, 10 -3 M, 10 -4 M and 10 -5 M.
[0039] (2) Before the injection begins, mussels that secrete byssus threads are selected from mussels that have been temporarily cultured for 1-2 weeks. The surface byssus threads are trimmed and placed in a glass dish. The mussels are temporarily cultured in a plastic basin containing 10 L of pumped seawater for 3 days to select mussels that secrete byssus threads for injection.
[0040] (3) Mussels that secrete byssus were selected from mussels that were temporarily cultured for 3 days and injected with adrenaline and serotonin. Neurotransmitter drugs were injected into the adductor muscle of thick-shelled mussels, 200 μL was injected into each mussel, and 21 mussels were injected into each concentration treatment group. After injection, the mussels were dried in the shade for 30 minutes and placed in pumped seawater. The number of byssus secreted by the mussels was counted at 6h, 12h, 24h, 48h and 72h after injection. The injection experiment set up a blank control group (Blank group) and a solvent control group (mussel saline, Vehicle group).
[0041] like Figure 3 As shown, 6h, 10-2 The amount of byssus secreted by the mussels in the MEPI injection group was significantly higher than that in the control group (P<0.05); 12-72h after injection, 10 -2 The amount of byssus secretion in the MEPI injection group was higher than that in the control group, and at 72h and 10h after injection, -3 The amount of byssus secreted by the MEPI-injected mussels was significantly higher than that in the control group (P<0.05). The experimental results showed that adrenaline may play a role in promoting the secretion of byssus by the thick-shelled mussels.
[0042] like Figure 4 As shown, 0-72h after injection of serotonin, 10 -2 ~10 -4 The amount of byssus secretion in the mussels treated with M concentration was lower than that in the control group. -5 There was no significant difference in the amount of mussel byssus secretion between the M concentration treatment group and the control group (P>0.05). The experimental results showed that the injection of 5-HT had no significant effect on promoting the secretion of mussel byssus (P>0.05).
[0043] The above analysis results show that by injecting 10 -3 After 72h of MEPI, the amount of byssus secreted by mussels was significantly higher than that in the control group (P<0.05), but 5-HT injection had no significant effect in promoting byssus secretion (P>0.05).
[0044] In summary, the present invention innovatively proposes a method for promoting the secretion of byssus in mussels from the perspective of neuroendocrine, which lays an important foundation for studying the secretion mechanism of mussel byssus and its physiological regulation effect.
[0045] This specific implementation is only an explanation of the present invention, not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims of the present invention.
Claims
1. The use of exogenous injected neurotransmitters to promote the secretion of mussel byssus, characterized in that: The exogenously injected neurotransmitter is adrenaline, and the application method is to inject adrenaline into the adductor muscle of the mussel to promote the secretion of its foot thread.