Use of olive oil to improve metamorphosis rate and / or survival rate of baby snails

Abstract

The application discloses application of olive oil in improving metamorphosis rate and / or survival rate of abalone larvae, wherein the volume concentration of the olive oil in abalone breeding water is 0.001-0.25%. In addition, the application also claims a method for promoting metamorphosis of abalone larvae, wherein olive oil is added into breeding water in which there are not-yet-metamorphosed abalone planktonic larvae, and cultivation is carried out; the volume concentration of the olive oil in the abalone breeding water is 0.001-0.25%. The olive oil provided by the application can greatly improve the metamorphosis rate and survival rate of the abalone larvae, shorten the planktonic period, and greatly improve the abalone breeding benefit.

Description

Technical Field

[0001] This invention belongs to the technical field of *Bellamya aegyptiacus* cultivation, and more specifically, relates to the application of olive oil in improving the metamorphosis rate and / or survival rate of *Bellamya aegyptiacus* larvae. Background Technology

[0002] In the past decade or so, significant progress has been made in the chemical induction of attachment and metamorphosis in marine gastropod larvae. Based on existing research, effective factors that can induce attachment and metamorphosis in gastropod larvae include natural inducing substances, neurotransmitters and their derivatives, and inorganic ions (K+). + As well as organic solvents. Because the metamorphosis mechanisms of different gastropod species vary considerably, it is necessary to examine and compare more species, especially newly studied gastropod species, to further elucidate the significance of these differences in larval attachment and metamorphosis mechanisms in population biology and systematic evolution.

[0003] The African whelk is an important marine biological resource, belonging to a type of marine gastropod larva. Due to its high economic value, the artificial cultivation of African whelks has developed rapidly. Currently, there are three main artificially cultivated species in my country: the spotted African whelk, the mud African whelk, and the Taiwan African whelk. Driven by market demand, the scale and area of ​​African whelk farming have been expanding year by year, making it one of the main species in southern marine aquaculture.

[0004] The paper "Artificial Breeding Technology of Spotted Warbler Snails" by Wang Guofu and the Hainan Provincial Fisheries Research Institute discloses an artificial breeding method for spotted warbler snails, including steps and considerations such as egg collection, egg hatching, larval rearing, feed selection, and larval rearing effects. The method describes a pre-metamorphosis survival rate of larvae in the snails, ranging from 64.6% to 70.8%, while the metamorphosis rate is generally between 10% and 64%, averaging 30.6%. Current snail breeding methods exhibit low larval metamorphosis rates with significant fluctuations. A lower metamorphosis rate means fewer snails undergoing metamorphosis and attachment, leading to a sharp decrease in the number of snails raised from metamorphosis to juvenile stage. This makes it difficult to meet the large market demand for snails.

[0005] "A Study on the Chemical Induction of Attachment and Metamorphosis of Two Types of East Wind Snail Larvae, Ke Caihuan, Department of Oceanography, Xiamen University" discloses the chemical induction of attachment and metamorphosis of two types of East Wind Snail larvae under continuous 24-hour exposure, with a concentration of 12 × 10⁻⁶ cells / day. -3 mol / dm 3 K + It can effectively induce complete metamorphosis in over 90% of the planktonic larvae of *Bellamya santalinus* and *Bellamya squarrosa*, while 15×10 -3 mol / dm 3 K + It has a toxic effect on the larvae of the spotted worm snail. Therefore, although a specific concentration of K... +Although it has a good effect on inducing metamorphosis in *Bellamya aegyptiacus*, different concentrations of K... + It can also be toxic to the East Wind Snail, and in recent years, K... + The induction effect of the spotted barnacle larvae is no longer satisfactory, with an induction rate often below 60%, which is no longer sufficient to induce the barnacles to settle on the bottom and metamorphose. The reason for this is still unknown, but it may be related to the excessive parasitic infestation of barnacles by substrate ciliates and other parasites.

[0006] The study, "Effects of Vitamin B7 and B12 on Bacterial Biofilm Formation and Metamorphosis of Thick-shelled Mussel Larvae," by Yang Jinlong (Shanghai Ocean University), disclosed that 0.02 and 0.2 mmol / L VB7 induced larval metamorphosis rates of (28.33±0.83)% and (11.67±0.83)%, respectively, while VB12 induced larval metamorphosis rates of (13.89±1.39)% and (1.67±0.83)%, respectively. The metamorphosis rates of 2 mmol / L and 20 mmol / L VB7 were (5.56±1.30)% and (1.67±0.83)%, respectively, while those of VB12 were (1.67±0.83)% and 0%, respectively. All these treatments showed significant inhibitory effects compared to the blank control group (P<0.05). However, the induction rates of the above treatments remained low. Therefore, improving the metamorphosis rate and survival rate of *Bellamya aegyptiacus* larvae has become an urgent technical problem to be solved. Summary of the Invention

[0007] To address the aforementioned technical problems, the primary objective of this invention is to provide the application of olive oil in improving the metamorphosis rate and / or survival rate of *Bambusa serpentina* larvae. The olive oil can significantly improve the metamorphosis rate and post-metamorphosis survival rate of *Bambusa serpentina* larvae, shorten the planktonic cycle, and greatly improve the seedling cultivation efficiency of *Bambusa serpentina*.

[0008] The second objective of this invention is to provide a method for promoting metamorphosis in the larvae of the Oriental Wind Snail.

[0009] The third objective of this invention is to provide a method for promoting metamorphosis in the larvae of the African worm snail and its application in increasing the metamorphosis rate and / or survival rate of the African worm snail larvae.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0011] This invention seeks protection for the use of olive oil in improving the metamorphosis rate and / or survival rate of *Bellamya aegyptiacus* larvae; wherein the volume concentration of the olive oil in the *Bellamya aegyptiacus* aquaculture water is 0.001–0.25%.

[0012] Through experimental research on various existing plant essential oils on the market, the inventors were surprised to find that olive oil, compared with other types of plant essential oils, has a superior effect on promoting the metamorphosis of Winged Snail larvae, and can greatly increase the metamorphosis rate and survival rate of Winged Snail larvae. In addition, the inventors also found through research that vitamins (B6, B7, B12) and potassium ions have limited effects on promoting the metamorphosis of Winged Snail larvae.

[0013] Preferably, the volume concentration of olive oil in the water used for Amur whelk farming is 0.001–0.05%. More preferably, the volume concentration of olive oil in the water used for Amur whelk farming is 0.005–0.01%. Most preferably, the volume concentration of olive oil in the water used for Amur whelk farming is 0.01%. Specifically, the volume concentration of olive oil can be 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, or 0.05%, and this application is not limited thereto.

[0014] Preferably, the olive oil is cosmetic olive oil. The cosmetic olive oil is commercially available.

[0015] Preferably, the density of the planktonic larvae of the East Wind Snail in the aquaculture water is 0.1 to 1 larvae / mL.

[0016] Furthermore, this invention claims protection for a method to promote the metamorphosis of *Bracharina gracilis* larvae, which involves adding olive oil to a culture water containing unmetamorphosed *Bracharina gracilis* planktonic larvae for cultivation; wherein the volume concentration of the olive oil in the *Bracharina gracilis* culture water is 0.001–0.25%.

[0017] Preferably, the volume concentration of olive oil in the water used for the cultivation of *Echinochloa crus-galli* is 0.001–0.05%.

[0018] Preferably, feed is added during the cultivation process. More preferably, the feed includes unicellular algae, microcapsules, shrimp chips, allicin, and multivitamins. More specifically, the unicellular algae are concentrated algae, including but not limited to seaweed, chaete algae, golden algae, and chlorella. More specifically, the concentration of the unicellular algae is above 1 million cells / mL.

[0019] More preferably, the dosage of the single-celled algae, microcapsules, shrimp chips, allicin, and compound vitamins per cubic meter of water is 30-40L, 5-6g, 3-4g, 0.3-0.8g, and 0.2-0.6g, respectively.

[0020] Preferably, the water body has a COD > 5 mg / L, a water temperature of 20–32°C, a pH of 8.4–8.6, and a salinity of 25–35.

[0021] Preferably, the cultivation time is 12 to 72 hours.

[0022] Preferably, during the cultivation period, 5g / m³ of Bacillus subtilis can be added daily. 3 .

[0023] Preferably, the aquaculture water is disinfected and filtered seawater. More specifically, ultraviolet disinfection and sterilization and filtration with a 0.1μm filter cartridge can be used.

[0024] Specifically, the *Babylonia* species are of the genus *Babylonia*, including but not limited to *Babylonia areolata*, *Babylonia lutosa*, or *Babylonia formosae*.

[0025] Furthermore, this invention seeks protection for the application of the above method in improving the metamorphosis rate and / or survival rate of *Bellamya spp.* larvae.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention provides the application of olive oil in improving the metamorphosis rate and / or survival rate of *Bracharina burmannii* larvae. The inventors were surprised to find through research that olive oil can greatly improve the metamorphosis rate and post-metamorphosis survival rate of *Bracharina burmannii* larvae, shorten the planktonic cycle, and greatly improve the seedling production efficiency of *Bracharina burmannii*. Attached Figure Description

[0028] Figure 1 A state diagram of the experimental barrel used in Example 1 to promote the metamorphosis process.

[0029] Figure 2 This is a diagram showing the state of the late-stage vegetative larvae in Example 1, which were just placed in the metamorphosis experimental bucket to promote metamorphosis.

[0030] Figure 3 The specimen is a juvenile Oriental Wind Snail that successfully metamorphosed after using olive oil in Example 1. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] The phrase "early vesicle larval stage" refers to the developmental stage in which two residual yolk sacs are clearly visible inside the larvae of the East Wind Snail, and the two vesicles are oval-shaped, symmetrical, and densely covered with cilia on their edges.

[0033] The phrase "mid-stage vesicle larva" refers to the period when the yolk sac of the East Wind Snail larva disappears and it develops distinctive features such as eyes, forelegs, a primary shell, and a pair of antennae.

[0034] The phrase "late vesicle larval stage" refers to the following: the larvae of the East Wind Snail have a snout on their heads, siphons, distinct gill filaments, and 1-2 rows of reddish-brown pigment spots on their whorls.

[0035] The phrase "juvenile snail" refers to the larvae of the East Wind Snail, whose facial discs shrink, cilia fall off, and they transition from planktonic to crawling.

[0036] The phrase "acidity / alkalinity" refers to the degree of acidity or alkalinity of a solution, usually expressed as pH value.

[0037] Example 1

[0038] (1) Inject seawater that has been sterilized by ultraviolet light and filtered by a 0.1-micron filter into the alternating tank, add olive oil (source: John Jeff brand produced by Shanghai John Jeff Cosmetics Co., Ltd., product name: olive oil extract), aerate and mix evenly, the volume concentration of olive oil in seawater is 0.001%, keep the COD of seawater > 5 mg / L, the water temperature is 27-29℃, the pH is 8.4-8.6, and the salinity is 29-30.

[0039] (2) The water level in the *Bellamya spp.* nursery pond was lowered, and planktonic larvae of *Bellamya spp.* that had not yet metamorphosed were collected using a 100-mesh silk screen. The larvae were washed twice with seawater and then placed in metamorphosis tanks. They were fed three times daily, with each feeding consisting of the following per cubic meter of water: 30L of concentrated seaweed (over 3 million cells / mL), 5g of microcapsules (source: Guangdong Yuequn Biotechnology Co., Ltd., trade name "Dongwan Miaobao"), 3g of shrimp chips (Taiwan Tongdi Company, Jiangkou brand, trade name "Grade A Shrimp Chips"), 0.5g of allicin, and 0.3g of compound vitamins (selected from Guangzhou Jinshui Animal Health Products Co., Ltd., trade name "Nutritional Express"). After 72 hours of cultivation, the planktonic larvae of *Bellamya spp.* successively progressed through the early vegetal larval stage, the middle vegetal larval stage, and the late vegetal larval stage. Finally, the metamorphosed *Bellamya spp.* juvenile snails were collected.

[0040] Example 2

[0041] The difference between this embodiment and Embodiment 1 is that the volume concentration of olive oil in seawater is 0.005%.

[0042] Example 3

[0043] The difference between this embodiment and Embodiment 1 is that the volume concentration of olive oil in seawater is 0.01%.

[0044] Example 4

[0045] The difference between this embodiment and Embodiment 1 is that the volume concentration of olive oil in seawater is 0.05%.

[0046] Example 5

[0047] The difference between this embodiment and Embodiment 1 is that the volume concentration of olive oil in seawater is 0.25%.

[0048] Example 6

[0049] The difference between this embodiment and Embodiment 1 is that the olive oil used is sourced from Herborist Biotechnology (Hong Kong) Limited, and is marketed as "Olive Oil Essence".

[0050] Comparative Example 1

[0051] The difference between this comparative example and Example 1 is that olive oil was not used.

[0052] Comparative Example 2

[0053] The difference between this comparative example and Example 1 is that vitamin B7 is used instead of olive oil, and the content of vitamin B7 in seawater is 0.02 mmol / L.

[0054] Comparative Example 3

[0055] The difference between this comparative example and Example 1 is that vitamin B6 is used instead of olive oil, and the content of vitamin B6 in seawater is 0.02 mmol / L.

[0056] Comparative Example 4

[0057] The difference between this comparative example and Example 1 is that vitamin B12 is used instead of olive oil, and the content of vitamin B12 in seawater is 0.02 mmol / L.

[0058] Comparative Example 5

[0059] The difference between this comparative example and Example 1 is that a compound vitamin (source: Guangzhou Jinshui Animal Health Products Co., Ltd., trade name "Nutritional Express") was used instead of olive oil, and the content of the compound vitamin in seawater was 0.02 mmol / L.

[0060] Comparative Example 6

[0061] The difference between this comparative example and Example 1 is that lavender essential oil (source: Yunnan Xicao Resources Development Co., Ltd., trade name "Mingshutang Lavender Single Essential Oil") is used instead of olive oil.

[0062] Comparative Example 7

[0063] The difference between this comparative example and Example 1 is that red ginseng essential oil (source: Herborist Biotechnology (Hong Kong) Limited, trade name "Red Ginseng Essence") is used instead of olive oil.

[0064] Comparative Example 8

[0065] The difference between this comparative example and Example 1 is that KCl is used instead of olive oil, and the concentration is set to 12 × 10⁻⁶. -3 mol / dm 3 .

[0066] Test Example 1

[0067] The metamorphosis rate and survival rate of *Bellamya spp.* during different time periods (12h, 24h, 48h, 72h) in the above embodiments and comparative examples were statistically analyzed.

[0068] Judgment criteria: Metamorphosis is considered complete when the vesicle of the East Wind Snail larva shrinks, ciliated cells fall off, loses the ability to swim, and can attach to the bottom or wall and crawl; during counting, the water is stirred counterclockwise or clockwise to create a rotating current, and the snails will gather in the middle of the bottom of the water. Successfully metamorphosed juvenile snails will crawl away, and those that do not move in the middle are dead juvenile snails or larvae; those floating in the water are vesicle larvae that have not successfully metamorphosed; the larvae that stop swimming, sink to the bottom, and no longer move are counted as dead; juvenile snails are considered alive if they can still crawl normally 48 hours after metamorphosis.

[0069] Metamorphosis rate = (Number of metamorphosed juvenile snails / Number of initial planktonic vesicle larvae in the later stage) × 100%;

[0070] Survival rate = (Number of live juvenile snails / Number of attached juvenile snails) × 100%;

[0071] The above statistical results are shown in Table 1 below.

[0072] Table 1

[0073]

[0074] The data above show that olive oil can significantly improve the metamorphosis rate and survival rate of *Bretschneidera sinensis* larvae. After 72 hours of treatment, the metamorphosis rate of *Bretschneidera sinensis* larvae was >61%, and the survival rate was >78%. More preferably, the metamorphosis rate of *Bretschneidera sinensis* larvae was >93%, and the survival rate was >95%.

[0075] As can be seen from Examples 1, 6 and Comparative Example 1, the use of olive oil can significantly improve the metamorphosis rate and survival rate of the late-stage vegetative disc larvae of the Oriental Wind Snail.

[0076] As demonstrated in Examples 1, 2, 3, 4, and 5, the olive oil of this invention significantly improves the metamorphosis rate and survival rate of *Bellamya aegypti* larvae compared to vitamins B6, B7, and B12. In contrast, various vitamins do not effectively promote metamorphosis in *Bellamya aegypti* larvae.

[0077] As can be seen from Examples 1, 6, and 7, the inventors, through experiments on various plant essential oils available on the market, were surprised to find that the olive oil of the present invention can significantly improve the metamorphosis rate and survival rate of the larvae of the African whelk, while other plant essential oils have limited effects on promoting the metamorphosis of the African whelk.

[0078] As can be seen from Example 1 and Comparative Example 8, 0.25% concentration olive oil and 12×10 -3 mol / dm 3 Compared to other concentrations of KCl, both had similar effects on promoting metamorphosis in *Bellamya aegyptiacus*.

[0079] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. The application of olive oil in improving the metamorphosis rate and / or survival rate of *Bellamya aegyptiacus* larvae, characterized in that, The volume concentration of olive oil in the water used for the cultivation of African whelks is 0.001–0.05%.

2. The application according to claim 1, characterized in that, The olive oil mentioned is cosmetic olive oil.

3. The application according to claim 1, characterized in that, The density of the *Bellamya spp.* larvae in the culture water was 0.1–1 larvae / mL.

4. A method for promoting metamorphosis in the larvae of the Oriental Wind Snail, characterized in that, Olive oil was added to the culture water containing unmetamorphosed planktonic larvae of the African whelk and cultured; the volume concentration of olive oil in the African whelk culture water was 0.001-0.05%.

5. The method according to claim 4, characterized in that, Feed is introduced during the cultivation process.

6. The method according to claim 4, characterized in that, The aquaculture water has a COD > 5 mg / L, a temperature of 20–32℃, a pH of 8.4–8.6, and a salinity of 25–35.

7. The method according to claim 4, characterized in that, The cultivation time is 12–72 hours.

8. The application of the method according to any one of claims 4 to 7 in improving the metamorphosis rate and / or survival rate of *Berberis thunbergii* larvae.

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