A tissue extract for inducing settlement metamorphosis of sea squirt larvae
By extracting and processing taurine-containing extracts from sea squirts or shellfish tissues, the lack of research on the attachment metamorphosis process of sea squirt larvae was addressed, enabling the induced attachment of sea squirt larvae and providing a research basis for the prevention and control of sea squirt fouling.
Patent Information
- Application Number
- CN202411931725.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-11-11
- Estimated Expiration
- 2044-12-26
AI Technical Summary
Existing technologies have not adequately studied the regulation mechanism of sea squirt attachment, making it difficult to effectively prevent and control the impact of sea squirt fouling on aquaculture, especially since the attachment metamorphosis process of sea squirt larvae has not been thoroughly understood.
A tissue extract from sea squirts or mollusks is provided, which, through grinding, centrifugation, low-temperature settling and precipitation, yields an extract containing taurine, which is used to induce attachment metamorphosis in sea squirt larvae.
The use of taurine can attract and promote the attachment metamorphosis of tunicate larvae, providing a basis for studying the chemosensory sensing system of tunicate larvae and providing clues for developing strategies to control tunicate attachment.
Smart Images
Figure CN119699234B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of amino acid substance function identification, specifically relating to a tissue extract that can induce attachment metamorphosis in sea squirt larvae. Background Technology
[0002] Sea squirts are diverse and widely distributed in all major oceans worldwide. They typically attach to various marine substrates and are typical invasive and fouling organisms globally. Research indicates that some important aquaculture species are primarily threatened by sea squirt fouling. Once sea squirts adhere to the surfaces of aquaculture facilities, they compete with farmed species for oxygen and nutrients, leading to decreased yields and severely impacting the aquaculture industry. In recent years, sea squirts have become the number one enemy of shellfish farming. In scallop and oyster farming, high densities of sea squirts attached to net cages and ropes are difficult to remove, and the annual cost of sea squirt control accounts for approximately 15% of the total aquaculture cost.
[0003] Research on the regulatory mechanism of tunicate attachment is key to understanding why tunicates have a strong invasive ability at the molecular level. However, the molecular mechanism regulating the attachment process is not yet fully understood. A deeper understanding of the mechanism of this process and the elucidation of important regulatory pathways and key functional proteins are urgent problems to be solved and are also an important foundation for developing and designing effective strategies to prevent and control tunicate attachment and fouling.
[0004] Sea squirts undergo a unique attachment metamorphosis process, evolving from swimming larvae to attached juveniles under the influence of various external and endogenous signals. Due to their immobility after attachment, the perception and selection of the external environment during the swimming stage are crucial. It is known that the external environment influences sea squirt metamorphosis; factors such as salinity, bacteria, and metal ions can affect the rate of metamorphosis. Sea squirts primarily perceive their environment through their peripheral nervous system (PNS). The PNS includes apical papillary neurons associated with the attachment site, rostral dry epidermal neurons (RTENs), head epidermal neurons (ATENs), dorsal and ventral caudal epidermal neurons (DCENs and VCENs), and terminal caudal neurons. During the swimming larval stage, sea squirts rely on different neurons in the PNS to perceive diverse external signals in order to find a suitable attachment site. Activation of these neurons, through neuroendocrine regulation (such as the action of neurotransmitters and neuropeptides), initiates the tail contraction process, rapidly undergoing metamorphosis to transform into a sessile adult. Therefore, the chemosensory and neuropeptide systems of sea squirts play an extremely important role in larval attachment and metamorphosis. Studying the chemosensory and neuroendocrine systems is an effective way to find key regulatory pathways and further develop anti-fouling biotechnology. Summary of the Invention
[0005] In view of the gaps in the existing technology mentioned above, and to study the attachment regulation mechanism of sea squirts and explore why sea squirts have a strong invasive ability, this invention discloses a tissue extract for inducing attachment metamorphosis in sea squirt larvae.
[0006] The present invention first provides a tissue extract for inducing attachment metamorphosis in sea squirt larvae, wherein the tissue extract is extracted from sea squirt or mollusk tissue.
[0007] Furthermore, the tissue extract is prepared by grinding sea squirt or shellfish tissue, centrifuging, and collecting the supernatant; adding trichloroacetic acid and allowing it to stand at low temperature; then centrifuging the supernatant; evaporating the supernatant after centrifugation, and dissolving the resulting precipitate in a solvent to obtain the tissue extract.
[0008] Furthermore, the aforementioned shellfish tissue refers to the adductor muscle, gonads, mantle, or gill filaments of a scallop.
[0009] Furthermore, the tissue extract contains taurine.
[0010] The present invention also provides an application of the tissue extract in inducing attachment metamorphosis in sea squirt larvae.
[0011] Another use of the tissue extract provided by this invention is in the preparation of products for inducing attachment metamorphosis in sea squirt larvae.
[0012] In another aspect, the present invention provides a method for inducing attachment metamorphosis in sea squirt larvae, the method comprising using the aforementioned tissue extract to induce attachment metamorphosis in sea squirt larvae.
[0013] Furthermore, the method involves replacing all or part of the tissue extract with taurine.
[0014] The beneficial effects of this invention are as follows: The applicant experimentally determined that extracts from shellfish and sea squirt tissues can attract sea squirt larvae for attachment. Subsequent mass spectrometry detection and screening of high-content components, along with experimental confirmation that taurine can attract sea squirt larvae for attachment metamorphosis, further demonstrated that taurine can attract sea squirt larvae for attachment metamorphosis. Taurine's ability to attract sea squirt larvae for attachment and promote tail contraction in sea squirt larvae can induce attachment metamorphosis. This discovery can be used to study the chemosensory system of sea squirt larvae, and further applied to combat sea squirt attachment by inhibiting the taurine chemosensory pathway in sea squirt larvae. Attached Figure Description
[0015] Figure 1 It is the structural formula of taurine;
[0016] Figure 2 This is a physical image of a chemotactic behavior experimental model, in which the slow-release block is highlighted by dye.
[0017] Figure 3 This is a graph showing the effect of model-based statistical analysis on the attraction of sea squirt larvae by extracts from various scallop tissues.
[0018] Figure 4 This is a partial example of the attraction effect of scallop muscle extract on sea squirt larvae, where bar represents 2 mm;
[0019] Figure 5 This is a graph showing the effect of using a model to statistically analyze the attraction of sea squirt larvae by sea squirt tissue extracts.
[0020] Figure 6 This is a partial example of the attraction effect of adult sea squirt extract on sea squirt larvae, where bar represents 2 mm;
[0021] Figure 7 This is a graph showing the results of using a model to statistically analyze the effects of various amino acids on the attraction of sea squirt larvae;
[0022] Figure 8 This is a graph showing the results of using a model to statistically analyze the attraction of different concentrations of taurine to sea squirt larvae;
[0023] Figure 9 This is a partial example of the attraction effect of taurine on sea squirt larvae, where bar represents 2 mm;
[0024] Figure 10 This is a statistical analysis of the results of taurine-induced tail contraction in sea squirt larvae using a model;
[0025] Figure 11 This is an example of how taurine promotes the contraction of the tail of sea squirt larvae, where bar represents 2 mm. Detailed Implementation
[0026] The present invention will be further described below with reference to the accompanying drawings and embodiments:
[0027] Example 1: Scallop tissue extract attracts sea squirt larvae to attach.
[0028] 1. Preparation of scallop tissue extract:
[0029] Scallop adductor muscle, gonad, mantle, and gill filaments were separately cut and minced in a mortar, liquid nitrogen was added, and they were thoroughly ground with a pestle. Approximately 0.5 g of tissue block was weighed into a lysis medium tube, 1 ml of PBS reagent was added, and the tissue block was lysed using a benchtop sample lysis instrument at 6.0 m / sec for 120 s, repeated twice. The tissue homogenate was transferred to a 2 ml enzyme-free centrifuge tube and centrifuged at 10000 x g for 15 min. The supernatant was collected, and an equal volume of 10% trichloroacetic acid was added. The mixture was allowed to stand at low temperature overnight to effectively precipitate proteins. The supernatant was then centrifuged at 12000 rpm for 30 min and evaporated in a 120°C metal bath until no obvious liquid remained. 200 μl of 1X HEPES solution was added to each tube, and the mixture was vortexed until no solids remained, yielding scallop adductor muscle, gonad, mantle, and gill filament tissue extracts.
[0030] 2. Construction of Chemotaxis Behavior Model
[0031] Establish a quantitative statistical model of chemical attraction to sea squirt larvae ( Figure 2 Its preparation method includes the following steps:
[0032] 1) Material preparation for model making: a plastic petri dish with a diameter of 60mm; a height of 8mm and a volume of 2cm³. 3 1) Container; a cylindrical tube with a diameter of 8 mm and smooth inner walls; agarose powder; a waterproof marker; seawater filtration. 2) Preparation of the slow-release block: A cylindrical tube with a height of 8 mm and a volume of 2 cm³... 3 1) Add tissue extract to the container and then add 2 ml of 1% agarose solution. After the agarose has completely solidified, cut it using a cylindrical tube with a diameter of 8 mm and smooth inner walls to obtain the slow-release block. 2) Statistical region delineation: Use a waterproof marker to draw a circle with a diameter of 40 mm on the outer surface of the bottom of the petri dish, concentric with the petri dish, and set it as the statistical region. 3) Fixing the slow-release block: Prepare a 1% agarose solution using filtered seawater. While the solution is still liquid, place it on the bottom of the petri dish and pour off any excess liquid. Before it solidifies, place the prepared slow-release block at the center of the circle and press it down manually to ensure it adheres to the bottom of the petri dish. 4) Model completion: Once the agarose at the bottom of the petri dish has solidified, immediately use a pipette to slowly add 5 ml of filtered seawater and gently shake the dish to observe whether the slow-release block has been completely fixed to the bottom. 6) Addition of experimental animals: Collect 200 swimming larvae of the tunicate that have good swimming ability. Use a 0.1% BSAcoat nozzle to prevent the larvae from sticking. Add the larvae evenly outside the statistical area in four batches using the treated nozzle.
[0033] 3. Observe and record the experimental results:
[0034] After adding larvae, the statistical area was photographed using a microscope every 30 minutes for 2 hours, i.e., observation time was 0 min, 30 min, 60 min, 90 min, and 120 min. After the experiment, the number of larvae in the photographs was counted, and the comparison parameter was set as the ratio of the total number of larvae in the statistical area to the total number of added larvae. A line graph was plotted using this parameter. Figure 3 , Figure 4 The results showed that among various tissue extracts, scallop muscle extract had the most significant effect on attracting choriotonic larvae to attach, followed by gonad extract, while gill filament and mantle extracts had no significant effect.
[0035] Example 2: Extracts from adult sea squirt tissue attract sea squirt larvae to attach.
[0036] 1. Preparation of extracts from adult sea squirts:
[0037] Adult, highly active *Sargassum salpinx* were collected, rinsed with filtered seawater, chopped in a mortar, and ground thoroughly with liquid nitrogen using a pestle. Approximately 0.5 g of tissue was weighed into a lysis medium tube, 1 ml of PBS was added, and the tissue was lysed using a benchtop sample lysis apparatus at 6.0 m / sec for 120 s, repeated twice. The tissue homogenate was then transferred to a 2 ml enzyme-free centrifuge tube and centrifuged at 10000 x g for 15 min. The supernatant was collected, and an equal volume of 10% trichloroacetic acid was added. The mixture was incubated overnight at low temperature to effectively precipitate proteins. The supernatant was then centrifuged at 12000 rpm for 30 min and evaporated in a 120°C metal bath until no obvious liquid remained. 200 μl of 1X HEPES solution was added to each tube, and the mixture was vortexed until no solids remained, yielding the tissue extract.
[0038] 2. Construction of Chemotaxis Behavior Model
[0039] The quantitative statistical chemotactic behavior model of the designed sea squirt larvae was prepared in the same way as in Example 1. Different concentrations (1x, 5x, 10x) of slow-release blocks were prepared by adding different concentrations of extracts, and concentration gradient behavioral experiments were conducted.
[0040] 3. Observe and record the experimental results:
[0041] After adding larvae, the statistical area was photographed using a microscope every 30 minutes for 2 hours, i.e., observation times were 0 min, 30 min, 60 min, 90 min, and 120 min. After the experiment, the number of larvae in the photographs was counted, and the comparison parameter was set as the ratio of the total number of larvae in the statistical area to the total number of added larvae. A line graph was plotted using this parameter. Figure 5 , Figure 6(This can represent the effect of chemical substances on the chemotactic behavior of sea squirt larvae over a period of time.) The results showed that extracts from adult sea squirt tissues could attract sea squirt larvae, and the attraction effect was concentration gradient dependent.
[0042] Example 3: Taurine attracts and induces attachment metamorphosis in sea squirt larvae
[0043] 1. Taurine attracts sea squirt larvae to attach.
[0044] Mass spectrometry analysis was performed on extracts from scallop muscle and sea squirt tissue. The results showed that the amino acids present in high amounts included Gly, Glu, Taurine, Arg, 5-HT, Phosphorylethanolamine (PE), Gln, and GSH (Tables 1 and 2).
[0045] Table 1. High-content amino acid substances in scallop muscle
[0046]
[0047] Table 2. High-content amino acid substances in sea tunicate tissue
[0048]
[0049] A 2.5 mM sustained-release compound was prepared using pure amino acids. A statistical model of chemotactic behavior in sea squirt larvae was used, prepared using the same method as in Example 1. After model creation, the number of larvae within the statistical area was continuously observed. Results showed ( Figure 7 Among various amino acids, taurine has a significant attraction effect on tunicate larvae, while the other substances have no significant effect on the chemotactic behavior of tunicate larvae.
[0050] To further confirm the absorption effect of taurine, taurine sustained-release blocks of different concentrations (500 μM, 1000 μM, and 2500 μM) were prepared, observed, and statistically analyzed to draw conclusions. Figure 8 , Figure 9 Within a certain range, the effect of taurine on attracting sea squirt larvae exhibits a concentration-dose effect.
[0051] 2. Taurine promotes metamorphosis in sea squirt larvae.
[0052] 2.1 A quantitative statistical model was established to investigate the effects of chemical substances on the tail contraction of tunicate larvae. The model was as follows: Using a 24-well cell culture plate, several morphologically normal and swimming tunicate larvae were collected. Then, 50 larvae and 500 μL of filtered seawater were added to each well. Different stimuli were then added according to the experimental plan, mixed well, and transferred to an 18℃ incubator for static culture. The larval morphology was observed under a microscope after 3 h, 6 h, 9 h, 12 h, and 18 h of treatment, and relevant data were recorded. Finally, the ratio of larvae that had completed tail contraction to the total number of larvae was compared after different treatments to compare the effects of each substance on the tail contraction of tunicate larvae.
[0053] 2.2 The above model was used to quantitatively and statistically analyze the effect of chemical substances on the tail contraction of sea squirt larvae. Different concentrations (10) of chemical substances were added to different wells. -5 M, 10 -6 M, 10 -7 Taurine (M) was added, and the tail contraction of larvae was observed after 3h, 6h, 9h, 12h, and 18h of culture. The proportion of larvae with completely contracted tails was also counted. The results showed that ( Figure 10 , Figure 11 As the concentration of taurine increases, the effect of promoting tail contraction in sea squirt larvae becomes more pronounced, especially at high concentrations of taurine (10). -5 After 9 hours of treatment with M), some larvae had completely shrunk their tails, while in the seawater control group, it took more than 12 hours for this phenomenon to occur.
[0054] The above results indicate that taurine, a sulfonic acid abundant in scallop and sea squirt tissues, can attract sea squirt larvae and promote tail contraction, thereby inducing attachment metamorphosis. This finding can be used to study the chemosensory system of sea squirt larvae, and further provide clues for anti-sea squirt attachment by inhibiting the taurine chemosensory pathway in sea squirt larvae.
[0055] The technical features of this invention not described can be implemented by or using existing technology, and will not be repeated here. Of course, the above description is not a limitation of this invention, and this invention is not limited to the examples above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention should also be within the protection scope of this invention.
Claims
1. A tissue extract for inducing attachment metamorphosis in sea squirt larvae, characterized in that: The tissue extract is extracted from shellfish tissue and contains taurine.
2. The tissue extract for inducing attachment metamorphosis in sea squirt larvae according to claim 1, characterized in that: The tissue extract is prepared by grinding shellfish tissue, centrifuging, and collecting the supernatant; adding trichloroacetic acid and allowing it to stand at low temperature; then centrifuging the supernatant; evaporating the supernatant after centrifugation, and dissolving the resulting precipitate in a solvent to obtain the tissue extract.
3. A tissue extract for inducing attachment metamorphosis in sea squirt larvae according to claim 1 or 2, characterized in that: The shellfish tissues mentioned refer to the adductor muscle, gonads, mantle, or gill filaments of scallops.
4. The use of the tissue extract as described in any one of claims 1 to 3 in inducing attachment metamorphosis in sea squirt larvae.
5. The use of the tissue extract as described in any one of claims 1 to 3 in the preparation of articles for inducing attachment metamorphosis in sea squirt larvae.
6. A method for inducing attachment metamorphosis in sea squirt larvae, characterized in that: The method involves using a tissue extract as described in any one of claims 1 to 3 to induce attachment metamorphosis in sea squirt larvae.
7. The method for inducing attachment metamorphosis in sea squirt larvae according to claim 6, characterized in that: The method involves replacing all or part of the tissue extract with taurine.