Tissue extract for inducing attachment and metamorphosis of sea squirt larvae

Abstract

The invention provides a tissue extract for inducing attachment and metamorphosis of sea squirt larvae, and belongs to the field of amino acid substance functions. Grinding sea squirt or shellfish tissues, centrifuging, and taking supernate; after trichloroacetic acid is added, low-temperature standing is carried out; then taking supernate and centrifuging; and evaporating the centrifuged supernatant, and dissolving the obtained precipitate in a solvent to obtain the tissue extract. The tissue extract can be used for inducing attachment and metamorphosis of sea squirt larvae.

Description

Technical Field

[0001] The invention belongs to the field of amino acid substance function identification, and in particular relates to a tissue extract capable of inducing the attachment and metamorphosis of ascidian larvae. Background Art

[0002] There are many species of sea squirts, which are widely distributed in major oceans around the world. They often attach to the surface of various substrates in the ocean and are typical invasive organisms and fouling organisms worldwide. Research surveys have shown that some important economic aquaculture species are mainly threatened by sea squirt fouling. After sea squirts adhere to the surface of aquaculture facilities, they will compete with farmed species for oxygen, nutrients, etc., resulting in a decline in aquaculture production, which has a serious negative impact on the aquaculture industry. In recent years, sea squirts have become the number one pest in shellfish farming. In the farming of scallops, oysters, etc., high-density sea squirts are attached to cages and ropes and are difficult to remove. The annual cost of sea squirt prevention and control accounts for about 15% of the total farming cost.

[0003] The study of the regulatory mechanism of tunicate attachment is the key to understanding at the molecular level why tunicates have strong invasive capabilities. However, the molecular mechanism regulating their attachment process is not well studied. In-depth understanding of the mechanism of this process and elucidation of important regulatory pathways and key functional proteins are urgent issues to be addressed. These are also important foundations for developing effective strategies to prevent and control tunicate attachment and fouling.

[0004] Sea squirts have a special developmental process of attachment metamorphosis, which is induced by a variety of external and endogenous signals from swimming larvae to juveniles after attachment metamorphosis. Since sea squirts cannot move after attachment, the perception and selection of the external environment during the swimming period is particularly important. It is currently known that the external environment has an impact on the metamorphosis of sea squirts, such as salinity, bacteria, metal ions and other factors that affect the metamorphosis speed. Sea squirts mainly rely on their peripheral nervous system (PNS) to perceive the external environment. PNS includes apical mastoid neurons, rostral trunk epidermal neurons (RTENs), head epidermal neurons (ATENs), dorsal and ventral caudal epidermal neurons (DCENs and VCENs) and tail terminal neurons related to the attachment site; in the swimming larval stage, sea squirts need to rely on different neurons in the PNS to perceive a variety of external signals in order to find a suitable attachment site. After the activation of the relevant neurons, the tail contraction process is initiated through neuroendocrine regulation (such as the action of neurotransmitters and neuropeptides), and the tail contraction process is rapidly metamorphosed to transform into an adult that lives a fixed life. Therefore, the chemical perception and neuropeptide system of tunicates plays an extremely important role in the larval attachment and metamorphosis process. Research on the chemical perception and neuroendocrine system is an effective way to find key regulatory pathways and further develop anti-attachment and fouling biotechnology. Summary of the invention

[0005] In view of the above-mentioned gaps in the prior art, the adhesion regulation mechanism of ascidian is studied to explore why ascidian has strong invasion ability. The present invention discloses a tissue extract for inducing adhesion and metamorphosis of ascidian larvae.

[0006] The present invention firstly provides a tissue extract for inducing the attachment and metamorphosis of ascidian larvae. The tissue extract is extracted from ascidian or shellfish tissues.

[0007] Furthermore, the tissue extract is prepared by grinding the ascidian or shellfish tissue and centrifuging it to obtain the supernatant; adding trichloroacetic acid and letting it stand at low temperature; then centrifuging the supernatant; evaporating the supernatant after centrifugation, and then adding a solvent to dissolve the precipitate to obtain the tissue extract.

[0008] Furthermore, the shellfish tissue is the adductor muscle, gonad, mantle or gill filament tissue of scallop.

[0009] Furthermore, the tissue extract contains taurine.

[0010] The present invention also provides a use of the tissue extract, which is the use in inducing the attachment and metamorphosis of ascidian larvae.

[0011] Another use of the tissue extract provided by the present invention is to prepare a product for inducing the attachment and metamorphosis of ascidian larvae.

[0012] In another aspect, the present invention provides a method for inducing the attachment and metamorphosis of ascidian larvae, wherein the method comprises using the tissue extract to induce the attachment and metamorphosis of ascidian larvae.

[0013] Furthermore, the method described herein is to use taurine to completely or partially replace the tissue extract.

[0014] The beneficial effects of the present invention are as follows: the applicant has experimentally determined that extracts from shellfish and ascidian tissues can attract ascidian larvae to attach. After mass spectrometry detection and screening of high-content components, it has been experimentally determined that taurine can attract ascidian larvae to attach and metamorphose. Taurine can attract ascidian larvae to attach, promote the contraction of the tail of the ascidian larvae, and then induce the ascidian larvae to attach and metamorphose. This discovery can be used to study the chemical perception system of ascidian larvae, and then be used to combat ascidian attachment by inhibiting the taurine chemical perception pathway of ascidian larvae. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the structural formula of taurine; Figure 2 This is a real-life picture of a chemotaxis behavior experimental model, in which the slow-release block is stained with a dye to highlight the effect; Figure 3This is a statistical result of the model used to show the effect of extracts from various tissues of scallops in attracting ascidian larvae; Figure 4 This is a partial example of the attraction effect of scallop muscle extract on ascidian larvae, where the bar is 2 mm; Figure 5 This is a statistical result of the model using the ascidian tissue extract to attract ascidian larvae; Figure 6 This is a partial example of the attraction effect of adult ascidian extract on ascidian larvae, where the bar is 2 mm; Figure 7 This is a statistical graph of the effects of various amino acids on attracting ascidian larvae using the model; Figure 8 This is a statistical model showing the results of using different concentrations of taurine to attract ascidian larvae; Fig. 9 This is a partial example of the attraction effect of taurine on ascidian larvae, where the bar is 2 mm; Fig.10 This is a graph showing the results of taurine-induced tail contraction of ascidian larvae using model statistics; Fig.11 This is an example of the effect of taurine in promoting the contraction of the tail of the tunicate larvae, where the bar is 2mm. DETAILED DESCRIPTION

[0016] The present invention will be further described below in conjunction with the accompanying drawings and embodiments: Example 1: Scallop tissue extract attracts sea squirt larvae to attach 1. Preparation of scallop tissue extract: Cut the adductor muscle, gonad, mantle and gill filaments of scallops into pieces in a mortar, add liquid nitrogen, and use a pestle to grind them thoroughly; weigh about 0.5g of tissue blocks into a lysis medium tube, add 1ml PBS reagent, and use a desktop sample lyser to break the tissue blocks, 6.0m / sec, 120s, and repeat twice. Pipette the tissue homogenate into a 2ml enzyme-free centrifuge tube and centrifuge at 10000xg for 15min. Take the supernatant, add an equal volume of 10% trichloroacetic acid, and let it stand overnight at low temperature to effectively precipitate the protein in the liquid; take the supernatant and centrifuge at 12000rpm for 30min, then evaporate it in a 120°C metal bath with the lid open until there is no obvious liquid. Add 200μl 1XHEPES solution to each tube, vortex and shake until there is no solid, and obtain scallop adductor muscle, gonad, mantle and gill filament tissue extracts respectively.

[0017] 2. Chemotaxis behavior model preparation A quantitative statistical model for the attraction of chemicals to sea squirt larvae was established ( Figure 2 ), the preparation method of which comprises the following steps: 1) Model making material preparation: plastic culture dish with a diameter of 60 mm, a height of 8 mm and a volume of 2 cm 3 Container; cylindrical tube with a diameter of 8 mm and a smooth inner wall; agarose powder; waterproof marker pen; filtered seawater. 2) Preparation of sustained-release block: In a volume of 2 cm with a height of 8 mm 3 After adding tissue extract to the container, add 2ml of 1% agarose solution. After the agarose is completely solidified, use a cylindrical tube with a diameter of 8mm and a smooth inner wall to cut it to obtain a slow-release block. 3) Delineation of statistical area: Use a waterproof marker pen to draw a circle with a diameter of 40mm on the outer surface of the bottom of the culture dish, which is concentric with the culture dish and set as the statistical area. 4) Fixation of slow-release block: Use filtered seawater to prepare 1% agarose solution, lay the bottom of the culture dish in a liquid state, and pour out the excess liquid. When it is not solidified, place the prepared slow-release block at the center of the circle and press it manually to ensure that it has adhered to the bottom of the culture dish. 5) Model completion: After the agarose at the bottom of the culture dish has solidified, use a pipette to slowly add 5ml of filtered seawater immediately, and shake it gently to observe whether the slow-release block has been completely fixed to the bottom. 6) Addition of experimental animals: Collect 200 swimming larvae of Ascidianus sabreii with good swimming ability, use 0.1% BSAcoat pipette tips to prevent the larvae from adhering, and use the treated pipette tips to add the larvae evenly outside the statistical area four times.

[0018] 3. Observe and record the experimental results: After adding the larvae, take photos of the statistical area using a microscope every 30 minutes for 2 hours, that is, the observation time is 0 min 30 min 60 min 90 min 120 min. After the experiment, count the number of larvae in the photos, set the comparison parameter as the ratio of the total number of larvae in the statistical area to the total number of larvae added, and draw a line graph with this parameter ( Figure 3 , Figure 4 ), which can indicate the effect of chemical substances on the chemotactic behavior of ascidian larvae over a period of time. The results showed that among various tissue extracts, scallop muscle extract had the most significant effect in attracting ascidian larvae to attach, followed by gonad extract, while gill filament and mantle extract had no significant attraction effect.

[0019] Example 2: Adult ascidian tissue extracts attract ascidian larvae to attach 1. Preparation of adult ascidian tissue extract: Take an adult of Ascidian sabinosum with good activity, rinse it with filtered seawater, cut it into pieces in a mortar, add liquid nitrogen, and use a pestle to grind it thoroughly; weigh about 0.5g of tissue blocks into a lysis medium tube, add 1ml PBS reagent, and use a desktop sample lyser to break up the tissue blocks, 6.0m / sec, 120s, and repeat twice. Pipette the tissue homogenate into a 2ml enzyme-free centrifuge tube and centrifuge it at 10000xg for 15min. Take the supernatant, add an equal volume of 10% trichloroacetic acid, and let it stand overnight at low temperature to effectively precipitate the protein; take the supernatant and centrifuge it at 12000rpm for 30min, then evaporate it in a 120°C metal bath with the lid open until there is no obvious liquid. Add 200μl 1X HEPES solution to each tube, vortex and shake until there is no solid to obtain tissue extract.

[0020] 2. Chemotaxis behavior model preparation The preparation method of the designed quantitative statistical chemotaxis behavior model of ascidian larvae is the same as that in Example 1. By adding different concentrations of extracts, slow-release blocks of different concentrations (1-fold, 5-fold, and 10-fold) are prepared, and concentration gradient behavior experiments are carried out.

[0021] 3. Observe and record the experimental results: After adding the larvae, take photos of the statistical area using a microscope every 30 minutes for 2 hours, that is, the observation time is 0 min, 30 min, 60 min, 90 min, and 120 min. After the experiment, count the number of larvae in the photos, set the comparison parameter as the ratio of the total number of larvae in the statistical area to the total number of larvae added, and draw a line graph with this parameter ( Figure 5 , Figure 6 ), which can represent the effect of chemical substances on the chemotactic behavior of ascidian larvae over a period of time. The results showed that adult ascidian tissue extracts can attract ascidian larvae, and the attraction effect is concentration gradient dependent.

[0022] Example 3: Taurine attracts and induces ascidian larvae to attach and metamorphose 1. Taurine attracts sea squirt larvae to attach The mass spectrometry results of scallop muscle and ascidian tissue extracts showed that the high-content amino acids included Gly, Glu, Taurine, Arg, 5-HT, Phosphorylethanolamine (PE), Gln, GSH, etc. (Table 1, Table 2).

[0023] Table 1 High-content amino acid substances in scallop muscle

[0024] Table 2 High-content amino acid substances in ascidian tissue

[0025] A 2.5 mM slow-release block was prepared using pure amino acids, and a statistical model of chemotaxis behavior of ascidian larvae was used. The preparation method was the same as in Example 1. After the model was completed, the number of larvae in the statistical area was continuously observed. The results showed ( Figure 7 ), among various amino acids, taurine has a significant attraction to ascidian larvae, while the other substances have no significant effect on the chemotactic behavior of ascidian larvae.

[0026] To further confirm the attraction effect of taurine, different concentrations (500μM, 1000μM, 2500μM) of taurine sustained-release blocks were prepared, and the conclusion was drawn after observation and statistics ( Figure 8 , Fig. 9 ): Within a certain range, there is a concentration-dose effect on the effect of taurine in attracting ascidian larvae to attach.

[0027] 2. Taurine promotes metamorphosis of ascidian larvae 2.1 A model for quantitatively analyzing the effects of chemical substances on tail contraction of ascidian larvae was established. The model was as follows: a 24-well cell culture plate was used. First, several Ascidian larvae with normal morphology and normal swimming ability were collected. Then, 50 larvae and 500 μL filtered seawater were added to each well. Different stimuli were added according to the experimental plan. After mixing, the mixture was transferred to an 18°C ​​incubator for static culture. The larval morphology was observed under a microscope 3 h, 6 h, 9 h, 12 h, and 18 h after treatment, and the relevant data were recorded. Finally, the ratio of larvae that had completed tail contraction to the total number of larvae after different treatments was compared to compare the effects of various substances on tail contraction of ascidian larvae.

[0028] 2.2 Using the above model to quantitatively calculate the effect of chemicals on the tail contraction of sea squirt larvae, different concentrations (10 -5 M, 10 -6 M, 10 -7 M) of taurine, and counted the contraction of the larvae's tails after 3h, 6h, 9h, 12h, and 18h ​​of culture, and counted the proportion of larvae with completely contracted tails to the total number. The results showed that ( Fig.10 , Fig.11 ), with the increase of taurine concentration, the effect of promoting the contraction of the tail of the sea squirt larvae became more obvious. -5 M) After 9 hours of treatment, the tails of some larvae had completely retracted, while it took more than 12 hours for this phenomenon to occur in the seawater control group.

[0029] The above results indicate that taurine, as a type of sulfoamino acid with a high content in scallop and sea squirt tissues, can attract sea squirt larvae and promote the contraction of the tail of sea squirt larvae, thereby inducing the attachment metamorphosis of sea squirt larvae. This discovery can be used to study the chemical perception system of sea squirt larvae, and then provide clues for anti-sea squirt attachment by inhibiting the taurine chemical perception pathway of sea squirt larvae.

[0030] Technical features not described in the present invention can be achieved through or by adopting existing technologies and will not be described in detail here. Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention should also fall within the scope of protection of the present invention.

Claims

1. A tissue extract for inducing attachment metamorphosis of ascidian larvae, characterized in that: The tissue extract is extracted from ascidian or shellfish tissue.

2. A tissue extract for inducing attachment metamorphosis of ascidian larvae according to claim 1, characterized in that: The tissue extract is prepared by grinding sea squirt or shellfish tissue and centrifuging it to obtain the supernatant; adding trichloroacetic acid and standing it at low temperature; then centrifuging the supernatant; evaporating the supernatant after centrifugation, and then adding a solvent to dissolve the obtained precipitate to obtain the tissue extract.

3. A tissue extract for inducing attachment metamorphosis of ascidian larvae according to claim 1 or 2, characterized in that: The shellfish tissue is the adductor muscle, gonad, mantle or gill filament tissue of the scallop.

4. The tissue extract for inducing attachment metamorphosis of ascidian larvae according to claim 1, characterized in that: The tissue extract contains taurine.

5. Use of any tissue extract as claimed in claims 1 to 4 in inducing settlement and metamorphosis of ascidian larvae.

6. Use of any tissue extract as claimed in claims 1 to 4 in the preparation of a product for inducing the attachment and metamorphosis of ascidian larvae.

7. A method for inducing the attachment and metamorphosis of ascidian larvae, characterized in that: The method is to use any tissue extract as described in claims 1 to 4 to induce the settlement metamorphosis of ascidian larvae.

8. A method for inducing attachment and metamorphosis of ascidian larvae according to claim 7, characterized in that: The method is to use taurine to completely or partially replace the tissue extract.

Images