JPS6232829A - Method for breeding gastropod, mollusc and other sea bottom motive animal - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for cultivating gastropod molluscs and other seabed and benthic motile animals, and particularly to a method for cultivating abalone.

[Background of the Invention II] Significant improvements in aquaculture of abalone and other gastropods have been made in U.S. Pat.
No. 0 and No. 4,253,418, and the disclosures of the above-mentioned patents are referred to and cited from time to time in this specification. The present invention relates to a novel method for growing large numbers of healthy abalone at the larval stage, where some bacteria and microorganisms can be fatal and at least one type of bacteria is essential.

The present invention improves the preparatory treatment prior to spawning of mature abalone, as well as purification, hatching, larval development, implantation, and post-implantation rearing. It discloses a method that allows a far greater number of living organisms to survive in Chizuki than before. These improvements include the use of sterile water at each growth stage. It also utilizes specific bacteria, each grown separately, to promote implantation, provide initial nutrition, and maintain water quality control.

Antibiotics are also used to inhibit the growth of unwanted bacteria. Yet another improvement is the addition of nutrients to the aquarium to promote the growth of the desired bacteria and make the antibiotics used less effective.

U.S. Pat. No. 4,183,322 discloses a method for cleaning, hatching, and developing larvae of freshly laid abalone eggs. It also explains spawning and fertilization, and teaches the following procedures to increase hatching and larval survival rates.

1. Eggs are separated from the seawater in which spawning takes place by a multistage screening method that utilizes seawater that has been filtered and treated to reduce the number of bacteria to about 100 cells/-ε or less.

2. Stemize the eggs in water with a bacterial count of 100 cells/mQ or less.

3. Raise the cape larvae in seawater filtered with a coarse filter that removes large components but retains biological components that control ammonia (passes particles of about 25 microns or less).

4. Maintain temperature control, dissolved oxygen levels, and approximate egg and larval densities depending on container construction.

This significantly reduces early mortality, with 90% of eggs hatching and 90% of early larvae surviving.

However, it was found that this high survival rate at very early stages was not maintained at later stages of growth. This increase in mortality rate is thought to be due to the invasion of wild pathogens from parent abalone into the cape formation tank and larval rearing tank, which then attack the organisms in their early stages.

US Pat. No. 4,226,210 describes the larval implantation and metamorphosis process and early nutrient uptake process in abalones and other benthic gastropods. The patent also states that during the ambulatory life of larvae for about 60 days after colony formation, there is a high mortality rate due to various factors, such as bacteria, the quality of the implantation surface, and the nature of the bacterial community present on the implantation surface. points out. Although the methods disclosed in this patent significantly reduce these deleterious effects and greatly improve survival rates compared to no measures taken at all, this patent still shows that mortality rates are significantly higher. admits.

The patent also points out that economically viable quantities of abalone can be planted and grown by employing a method that includes the following steps:

1. Condition the implantation surface in advance to promote implantation and hasten nutritional intake by growing bacterial communities on the implantation surface.

2. Use seawater that contains no or almost no ingredients harmful to young abalone.

3. Maintain proper water quality by controlling biological balance by providing nutrients through photosynthesis by specific algae and addition of plant nutrients, and by controlling waste products.

4. Controlling temperature, oxygen levels, larval concentration, water circulation, and other factors.

In a preferred embodiment disclosed in U.S. Pat. No. 4.26f3,210, coastal seawater is filtered to remove large components, while relatively small
By leaving wild microorganisms of 0 micron or less in size and sending this water to the implantation tank for several days to flow over the implantation surface, a bacterial colony that promotes implantation is formed. This patent also describes the use of (1) the use of seawater free of harmful components obtained through processes such as preliminary filtration, and (2) the use of separately cultured microorganisms such as bacteria to condition the implantation surface. Suggests.

The technology disclosed in this patent has significantly improved aquaculture production, but
It is desired to further reduce mortality rates.

The purpose of the invention is to precisely control all biological processes in order to prevent the invasion and proliferation of harmful bacteria and other harmful biological components, while increasing the implantation rate and speeding up nutrient uptake. , eggs, larvae and subsequent growth stages by providing sufficient bacteria or other microorganisms in quality and quantity through the controlled culture process to achieve high survival rates and growth rates for several months after colonization. The aim is to improve the technology for cultivating abalone.

Both of the above-mentioned patents point out the harmful effects of bacteria on eggs, larvae and subsequent stages of development.
US Pat. No. 4,183,322 proposes using bacterial-sized wild components to control ammonia. Similarly, U.S. Pat. No. 4,226,210 describes the use of wild bacteria and similar-sized biological components to precondition implantation surfaces to promote implantation and speed up nutrient uptake. is suggesting. Wild bacteria numbers are allowed up to about 100 cells/le, in a 2 million liter tank commonly used for colonization, larval rearing or implantation, as described in U.S. Pat. No. 4,253,418. If necessary, a total of 2×10 8 bacteria can be introduced at each stage. However, the introduction of such a large number of microorganisms may also introduce harmful pathogens, which may significantly increase the mortality rate in the later stages of growth.

The present inventors have discovered that the survival rate of larval abalones for 60 days after grouping can be significantly improved by using antibiotics, predetermined bacteria cultured separately, and sterile seawater.

The method of the invention largely eliminates the possibility of introducing harmful organisms, provides the necessary bacteria grown separately, and provides antibiotics to control unwanted pathogens should they be introduced. do.

It is known that larval abalones require bacteria in their surrounding environment in order to consume ammonia in the later stages of larval life. In carrying out the present invention, the predominant bacteria that consume ammonia during the larval stage are first separated from wild bacteria, and implantation is promoted for larval metamorphosis and nutrients are provided immediately after implantation. At this stage, various bacteria are isolated from natural, unfiltered seawater using standard procedures employed for the isolation and cultivation of marine bacteria.

The next step is to test each isolated pure culture. The culture solution cultured for 24 to 48 hours was diluted with seawater filtered through a 0.2μ membrane, and then diluted with 100ml of filter-sterilized water.
I! was added to the sterilizer containing the 5 in each of 5 identical bowls containing each diluted culture to be tested.
Add 0-100 swimming larvae. After adding the larvae and bacteria, the abalones are placed in an environment at an appropriate temperature depending on the type, and the number of settlements, deaths, and numbers still in a swimming state are monitored daily.

After about 30 days, the results of this screening reveal pathogenic bacteria, non-affective bacteria, and bacteria that promote implantation and support rapid growth during the post-implantation period. The predominant bacteria are then maintained in culture using standard bacteriological techniques. The selective isolation, testing, cultivation and utilization of such preferred bacteria is an important aspect of the present invention.

Bacteria that we have found to have the necessary beneficial effects have the following common properties:

(1) A single carbon compound can be used as an energy source and a growth element, and ammonia can be used as a sole nitrogen source.

(2) Does not require growth factors such as vitamins.

(3) Respiratory metabolism is different from fermentative metabolism.

(4) does not produce toxic metabolic products or release more complex toxic substances;

The selected bacterium was Shutomonas cepacia (P5e
LIdOIIIonaS Ce1)aCia) and Pseudomonas m.
arginata), which will be described in detail later.

It is also an important component of the present invention to select and culture preferred bacteria that proliferate and grow in the presence of the preferred antibiotic. To prevent such resistant bacteria, appropriate antibiotics should be used to suppress harmful bacteria, and water quality should be controlled at the same time.
It is useful in promoting implantation and providing nutrients necessary to sustain life during early growth.

It is also a point of the invention to select and cultivate preferred bacteria that are resistant to naturally occurring bacteriophages that can enter through the filter and kill selected bacteria in a short period of time. be. Still another point is that the present invention involves the addition of chemical compounds that feed these desirable bacteria and, due to their growth-promoting properties, increase the sensitivity of undesirable bacteria and make antibiotics more effective. be. More details on how to implement these aspects of the invention are provided below.

It has been demonstrated that the improvements disclosed herein can eliminate or suppress harmful pathogens and eliminate the largest cause of death during abalone growth after two months of turbulence. When using coastal seawater as in the past, if this seawater is not filtered, the number of wild bacteria is 10,000 cells/Cm3 (mQ
) may also be reached.

Even with the filtration and sterilization treatments described in the above-mentioned patent, although the total number of wild bacteria is a low percentage, the number of pathogenic bacteria can cause the development of disease in young abalone and cause mass mortality over the next 60 days. There may be enough to bring it about. Therefore,
We have developed a method to completely or almost completely eliminate this wild fungus.

The ideal way to exterminate disease-causing bacteria is to eliminate all wild bacteria from the aquaculture process, but it is virtually impossible to achieve this ideal in actual production. Although it is possible to treat seawater to eliminate all bacteria,
Although it is possible to sterilize tanks, equipment, and equipment using heat, chemicals, and other means, abalone eggs still retain the bacteria they acquired from their mothers during spawning. At present, there is no way to obtain sterile eggs from abalone mothers. Therefore,
Pathogens or potential pathogens are always present, and the only option is to destroy or suppress them so that they do not proliferate in numbers that would increase the mortality rate of abalone after the larval stage.

During the process of developing this method, it was determined that the main pathogen was marine Durum-negative bacteria. Furthermore, polymyxin B
It has also been found that antibiotics such as 1 chloramphenicol, ampicillin, erythromycin, and chlortetracycline are extremely effective in eliminating or inhibiting the growth of such pathogenic bacteria.

Although other antibiotics, such as neomycin, streptomycin, zentamycin, etc., are also effective, it is preferable to use one or more of the compounds belonging to the former group in the practice of this invention. However, the invention is not limited to the use of these compounds.

[Summary of the Invention] The method of the present invention involves pre-treating adult males and females with antibiotics prior to spawning, which can serve as a dangerous source of undesirable bacteria during subsequent rearing stages and provide nutrients for bacterial growth. , to eliminate organic substances such as excess sperm, excrement, mucus, etc.
The eggs are filtered from the spawning and fertilization water according to the method of No. 3,322. Carefully separated eggs are transferred to a nearly sterile colony seawater tank containing one or more antibiotics. The eggs remain in the incubator for the nail egg stage until most of the grouped larvae leave their respective residual egg sacs. The mature larvae are transferred to a new, nearly sterile seawater tank containing one or two antibiotics, and during this transition stage the larvae are carefully separated from the colony residue. A multi-bath system that combines several antibiotics with the stepwise separation of eggs and larvae from the residue (as described in U.S. Pat. No. 4,183,322) significantly increases survival rates. However, it was found that the overall health condition of the abalone after implantation was much improved compared to conventional methods.

[Preferred Embodiment] An effective method of the present invention for hatching a large number of eggs and cultivating larval and post-larval abalone throughout its larval stage involves processing adult abalone prior to spawning, and treating all stages of abalone. It is a multi-step method that controls the quality of the seawater used for production, carefully processes eggs, larvae and post-larval abalone, and controls microbial populations throughout these stages of development.

Although there are many methods for obtaining sterile seawater, one of two methods is used in carrying out the present invention. The first preferred method is a sand filter, two stages of diatom ± filter, two stages of ultraviolet irradiation, two stages of spinning taxi I (selected to exclude particles larger than about 1.0 and 0.5 μ, respectively). fiber cartridge filtration, and as the final stage, a membrane filter that can exclude all particles larger than 0.2μ (commercially available filters include MF-M1
llipore Fitter's GS type (0,2
2μM) and [) GV of uraporeFilter
type (preferably 0.22 μM>) to filter coastal seawater over a wide area. This method eliminates almost all bacteria. However, pathogenic bacteria smaller than 0.2μ cannot be detected. A coarse filter of 0.5 μ or more may be used, but for safety reasons, 0.2
μ filters are preferred.

The second preferred seawater purification method is to collect seawater from the deep sea at least 1,000 feet deep, and to reduce the number of bacteria to 100 microns/ra.
(1) In most cases, less than 1 cell/rthQ (1) Cold seawater is used, and all of these bacteria can be eliminated by one filtration with a 0.2μ membrane filter.

Using highly filtered seawater, the bacteria that affect abalone eggs, larvae, and post-larval abalone can be extracted from an estimated 2 x 108 wild fungal cells (contained in the large tank above) at each stage of growth. It is almost limited to bacteria that are transferred from adults to eggs and subsequent larvae and continue to be present.

Treating adult male and female abalones with antibiotics prior to spawning reduces contamination of eggs and sperm by harmful adult-associated bacteria during the process from spawning to implantation. Methods of promoting spawning and fertilization are not included in the present invention. US Patent No. 4
, 183, 322, at the same time that spawning occurs, the eggs are cleaned containing egg sac residue, mucus, and other contaminants, including free or attached bacteria from the adults. Minutes from contaminated water! Do 1. This separation is accomplished by multiple filtration, which removes the eggs from contaminated water and transfers them to water that has been treated to sterile conditions. Sperm, carefully separated from egg sac residue and mucus, are then added, and once cell separation has begun, fertilized eggs are separated from excess sperm.

In natural breeding grounds, only 10 eggs survive this stage.
It is thought that it is only a few in 10,000. The aim of the present invention is to significantly increase survival numbers by the controlled method described herein. Since the above patent was issued, survival rates of fertilized eggs and early postlarval stages have significantly improved. When using seawater that has undergone final filtration with a filter that removes components larger than 0.2μ, remaining bacteria are almost completely removed. However, the surface of the fertilized egg has bacteria that cannot be removed by physical means such as filtration or washing with nearly sterile seawater. Treatment of adults before spawning helps to reduce sources of contamination. Adding one or more antibiotics to the nearly sterile water containing the eggs during colony formation is effective in inhibiting bacterial growth. Bacterial growth is further inhibited by the use of one or more antibiotics in the water in which the swimming larvae are transferred. If larvae are treated with antibiotics during the implantation and post-implantation periods, this suppressive effect on wild pathogens can be maintained for a long time.

As described in U.S. Pat. No. 4,226,210, since larvae are sensitive to their choice of landing surface, we further improved the method of preparing the landing surface disclosed in that patent. . Thus, unlike the known methods of growing wild bacteria and other microorganisms on the implantation surface in aquaculture systems, it has been found that the use of separately cultured specific bacteria provides much better results.

Another improvement of the present invention is that it is made resistant to certain antibiotics to encourage implantation and provide early nutrition in a bath that also contains antibiotics used to control harmful bacteria in the wild. It consists in using selected pure species of preferred bacteria. Selection of bacteria resistant to antibiotics is accomplished by repeated subculturing with increasing concentrations of antibiotic using standard bacteriological techniques until sufficient resistant bacteria are obtained. .

A further improvement of the present invention consists in the use of antibiotic resistant bacteria selected to be resistant to bacteriophages that are harmful to the selected bacteria.

Bacteriophage-resistant bacteria can be obtained by treating a large number of bacteria with bacteriophages, repeating subculturing, and then isolating bacteria that do not show any tendency to support the growth of destructive or lytic bacteriophages. Bacteriophages are often present in seawater and cannot be removed by filtration with a 0.2μ filter. In addition to the above-mentioned methods, it is thought that it is possible to develop even better bacteria through genetic manipulation, although this is still an undeveloped field.

The currently preferred beneficial bacteria is Pseudomonas cepacia.
and P seudomonas v.
nas marginata), R, E
, Bachnan and N.

Co-edited by E., G. 1bbins, Williams & Wilkins, Baltimore, 1914.
“3ergey =s M” issued by Co.
annual of [)etera+1nativ
Part of this is explained in e3acteriology u 8th edition. A more complete description of these bacteria can be found in J.

Gen Microbiol, (1966), 4
3. R, Y, 5ta published in 159-271
iner, N. J., Pa1ler.

nl and M, Doudoroff's "The A"
erobic p seudog+onads:
A T axonosic S tudy” and J.

Gen Microbiol, (1970), 6
0. R, W, Ba1 published in 199-214
lard, N. J., Pa1ler.

ni, M., Doudoroff, R.Y.
3tainer and M.

Mandel's “Taxonomy or the A
Erobic Pseudosands: Pse
udosonas cepacia, P, s
arginata, P. alliicola, and P. caryophyl ii.

The bacterium in question appears to be related to these Xutomonats, but does not exactly match any of the species described here.

The beneficial bacteria in question are A.
(:, A during culture Co11ection
T CCN o, number t' P seu of 39451
domonas sp, M onterey A
It is named balone Far@S3 tratn 13. This bacterium has the following characteristics:

(+) It is a Durham-negative rod-shaped bacterium that moves by the movement of multiple flagella at both poles.

(Ri) Metabolism is not fermentative metabolism but respiratory metabolism, and nitrate cannot be reduced to nitrite, nor can it live under anaerobic conditions due to denitrification.

(3) Does not fix nitrogen, is catalase positive, oxidase reaction is weak to negative, and does not produce indole.

(4) Storing poly-β-oxybutyrate as a cell reserve.

■ Forms a yellow non-fluorescent pigment in certain media.

(Q Does not produce arginine dihydrolase and does not produce extracellular hydrolases for gelatin or starch.

■ It does not require any particular growth element and can grow on ammonia or nitrate as a nitrogen source.

■ Any of the following compounds can be used as the sole source of carbon and energy. D-lipose, D-glucose, D-mannose, D-galactose, D-fructose, sucrose, trehalose, maltose, gluconate, vinegar M salt, propionate, butyrate, malonate, succinate, DL - malate, DL-β-hydroxybutyrate, DL-lactate, citrate, pyruvate, mannitol, meso-inositol, glycerol,
Ethanol, n-propertool, glycine, L-α-alanine, β-alanine, cycloserine, L-leucine, cycloisoleucine, L-aspartate, L-glutamate, L-lysine, cycloarginine, -ornithine, L-tyrosine, λ-aminobutyrate, L-proline, -7-enylalanine, betaine, and asparagine.

■ None of the following compounds can be used as the sole source of carbon and energy. D-xylose, D-arabinose, L-arapinose, D-7 course, L-rhamnose, cellobiose, lactose, starch, 2-
Ketogluconate, caproate, pelargonate, oxalate, maleate, adipate, DL-tartrate, glycolate, levulinate, sorbitol, ethylene glycol, dulcitol, methanol, isopropanol , n-butanol, geraniol, benzoate, p-hydroxybenzoate, m-hydroxybenzoate, phenylacetate, phenol, testosterone, L-threonine, DL-norleucine, L-
Valine, δ-aminovalerate, L-Si1~lurin, L
- Histidine, L-tryptophan, p-aminobenzoate, putrescine, histamine, creatine, pantothenate, acetamide, and nicotinate.

Addition of chemical nutrients to the seawater contained in the implantation tank is effective in maintaining viable populations of selected beneficial bacteria.

This bacterial growth is necessary to form a suitable implantation surface, provide sufficient beneficial bacteria as initial nutrients, and limit the concentration of harmful components such as ammonia released from larval metabolism. .

Addition of nutritional bacteria during early antibiotic treatment may also result in increased efficacy of the antibiotic against harmful bacteria that may become resistant, even if growth is temporarily stopped.

2 to 4 parts/1,000,00 to seawater tank containing introduced bacteria
Addition of 0 parts per million (ppm) of sodium citrate was found to promote the growth of selected bacteria.

Although the addition of this specific nutrient is preferred due to its role in the nutritional uptake of specific bacteria, the invention is not limited to the addition of this specific compound, and other nutrients such as sodium acetate, glucose, sucrose, etc. You can also use things.

Select nutrients by considering the following points: Nutrients are easily metabolized by most beneficial and harmful bacteria;
Moreover, this nutrient must not contain nitrogen. This is because nutrient metabolism for growth by beneficial bacteria takes up and removes toxic ammonia, which is used by the beneficial bacteria as a nitrogen source.

To explain more specifically from the examples, the present inventors have developed a method in which adults are treated prior to spawning, and eggs, larvae, and abalone immediately after the larval stage are raised in a bacteriologically controlled environment. It is. A preferred method includes the following steps.

a. Treat pre-cleaned adult abalone (with a brush) with one or more antibiotics for at least 48 hours prior to spawning, or for a period sufficient to reduce levels of harmful marine bacteria associated with the adult abalone. The substance is contained in substantially sterile seawater.

b. Replace antibiotic-containing seawater with nearly sterile seawater. Egg laying, fertilization, and separation of fertilized eggs are performed using the method described in US Pat. No. 4,183,322.

c. The separated and washed fertilized eggs are stored in a second seawater tank in a substantially sterile condition containing one or more types of antibiotics for a period required for nailing.

d. U.S. Pat. No. 4, U.S. Pat.
No. 183,322, selectively transferred to a third sea tank under substantially sterile conditions containing one or more antibiotics. Add bacteria.

e. Transfer the grown larvae from the third sea tank to a nearly sterile (>S bed sea tank as described in U.S. Pat. No. 4,253,418) and place one or two species in this settlement tank. Add antibiotics.

At the same time, it promotes the implantation of swimming larvae, provides food for the growth of abalone after implantation and metamorphosis, metabolizes ammonia released through the metabolism of larvae, and produces a separate product that is resistant to antibiotics. Add cultured and selected beneficial bacteria.

Preferred antibiotics used alone or in combination in this method are polymyxin B, erythromycin, ampicillin, chloramphenicol and chlortetracycline. It is common to use active antibiotics at a concentration level of 10 ppm dissolved in seawater, but concentrations can be above or below this level, and the method of the invention is not limited to the specific antibiotics and concentration levels mentioned above. do not have.

In the method of the present invention, one or more antibiotics are used as shown in 2 below.

a. Using one type of antibiotic in any one or more of the above steps.

b. Using one or more antibiotics in any one or more of the above steps.

c. Using a series of antibiotics in more than one step.

The antibiotic to be used is selected based on its effectiveness in preventing death due to harmful bacteria and non-toxicity at effective concentrations. For example, chloramphenicol, tetracycline,
Antibiotics such as novobiocin are non-toxic to swimming larvae but toxic to eggs, so they cannot be used at the egg stage. The use of a range of antibiotics increases the range of action of the antibiotics and thus effectively suppresses a variety of harmful bacteria that may be present in different larval tanks.

In a preferred embodiment of the invention, a battery of antibiotics is used throughout the treatment of the adult until 30 days after placement of the larva in the implantation tank. Another improvement to this method is that favorable bacteria survive in the presence of a specific antibiotic or combination of antibiotics, while harmful bacteria that inadvertently invade are cultured separately so that they are killed or suppressed by the antibiotic. Use antibiotic-resistant beneficial bacteria. Yet another improvement is the use of separate cultures of batataeriophage-resistant and antibiotic-resistant beneficial bacteria.

In the practice of the present invention, bacterial nutrients are added to the seawater to maintain and grow the population of selected beneficial bacteria in the implantation tank. Preferred nutrients are Illll-
41) Sodium citrate at pm concentration.

Specifically, male and female abalones are housed in separate approximately 50e spawning tanks 48 hours before expected spawning. Thoroughly wash the abalone shells with a brush to remove the abalone colonies that may contain bacteria. The water in the tank is either nearly sterile or filtered to keep the number of bacteria extremely low.

In this example, the antibiotic chlortetracycline is added to the bath water and dripped into the influent stream to maintain an initial level of active antibiotic, e.g.
000m. The tank is then drained and replaced with sterile water for the larvae to spawn. After the eggs are captured with a 100μ screen, they are passed through a 300μ screen at each subsequent step to eliminate mucus, egg sacs, and other undesirable substances, and the thus filtered and washed eggs are sterilized and washed again. Then transfer to grouping tank.

In most cases, several hundred and five eggs are produced, so a large hatching tank is required. If the number of eggs is like this, it will be about 2000.
Disinfect the H tank with chlorine in advance, air dry it, and
It is filled with almost sterile seawater that has been filtered through a μ filter, and the eggs are housed in it. A second antibiotic is then added to this second tank. In this example, the second antibiotic is at a concentration of 10 p
pm polymyxin B.

After a few days, the eggs cluster into free-swimming larvae, which are collected using a 100μ screen and gently washed with sterile water.
2000Q filled with almost sterile seawater that has been chlorinated, air-dried, and filtered through a 0.2μ filter.
Transfer to the third tank. A third antibiotic, ampicillin in this example, is added at a concentration of 10 pp-. Gently mix the larvae with aeration to maintain adequate oxygen levels with ampicillin-resistant milk. It was found that a larval density of 10 larvae/at or 20 larvae/1ffi could be sufficiently raised in this culture system.

Red abalone ()-1aliotis rufescens
), the larvae become ready for implantation after being housed in these seawater tanks whose temperature is controlled not to exceed 15°C for about 48 hours. It was then collected through a 100 μ screen, gently washed with sterile water, and then collected using a 100μ screen, washed gently with sterile water and
2000Q containing multiple faces as described in No. 8
Transfer to i1 bed tank. These implantation surfaces and the tank itself were sterilized in advance with dissolved chlorine at a concentration of about 10 (18) 11) times, air dried, and then filled with seawater filtered through a 0.2μ filter.

A fourth antibiotic, chloramphenicol in this example, is dissolved in seawater at a level of 10 ppm. Preferred bacteria selected to be resistant not only to the antibiotic chloramphenicol but also to bacteriophages and grown separately in pure culture (e.g. ATCC No. 39451 mentioned above)
) Add a culture containing approximately 5xlo11 cells. Mix the contents of the tank by slowly venting. Furthermore, 1
Bacterial nutrients consisting of 0g of sodium citrate are added to grow the necessary bacteria while also enhancing the inhibitory and bactericidal effects of antibiotics on wild bacteria that have persisted through the aquaculture process from adulthood or that have entered at any stage. enhance

In most cases, abalone larvae become young gastropods that crawl on the water bottom about 14 to 21 days after settling. The contents of the implantation tank remain unchanged for approximately 45 days, even with the addition of selected antibiotic and bacteriophage resistant cultures as needed. Whether or not the addition of the cultured bacteria is necessary is determined once a week by smearing a cotton swab sample of the implantation surface onto an agar plate.

Approximately 14 days after introducing the larvae into the final settlement tank, the organisms in the tank can be reduced by adding the diatom Navicula to the tank, adding diatom nutrients, and shining ambient light above the water surface into the tank. As well as controlling the biological and chemical balance, young abalones continue their metamorphosis and promote photosynthesis, providing another food source as they enter adolescence. Methods for cultivating seed diatoms under sterile conditions are known to those skilled in the art.

In this example, the present inventors found that more than 60% of the larvae housed in the implantation tank were viable, whereas conventionally 20% was considered a reasonable survival rate. . This represents a significant improvement both over the natural case and over known aquaculture. Although this example relates to red abalone, the method of the invention can be applied to other types of abalone, such as pink abalone Haliotis corrugata.
), other gastropod molluscs, such as conch, loco (
loco), and other seabed and benthic motile animals, such as sea urchins, which are economically and academically important. The method of the present invention is also believed to be particularly useful for culturing sea urchins, which are commonly found in nature in environments favored by abalone.

Claims (25)

[Claims] (1) A method for growing gastropod molluscs and other submarine and benthic motile animals in a microbiologically controlled environment for the first few months, a. Almost sterile bacteria containing antibiotics; House the cleaned adult animal in a controlled seawater tank, b. Collect eggs and sperm from the adult animal, and house the fertilized eggs in an almost sterile hatching seawater tank containing antibiotics; c. Incubate the hatched larvae. removed from the tank and immersed in a nearly sterile breeding seawater tank containing an antibiotic, d. 1. A method for cultivating gastropod molluscs and other benthic and benthic motile animals, which comprises the step of transferring developing larvae to a seawater tank under a controlled condition. (2) Adult animals are housed in a bacterial control tank for about two days before spawning, and then the tank is drained and refilled with fresh, nearly sterile seawater, and the animals are allowed to spawn in this seawater. The method described in section (1). (3) The growth tank also contains certain beneficial bacteria that are resistant to antibiotics in the growth tank.
The method described in section 1). (4) The method according to claim (1), wherein the hatched larvae are housed in a growth tank for at least one day. (5) The method according to claim (1), wherein the beneficial bacteria are separately cultured and selected to be resistant to bacteriophages. (6) The method according to claim (1), wherein bacterial nutrients are added to the implantation tank. (7) The method according to claim (6), wherein the nutrient is sodium citrate. (8) Beneficial bacteria a. can use a single carbon compound for energy and growth and can use ammonia as the sole nitrogen source; b. do not require growth factors such as vitamins, etc.; The method according to claim 7, which has the following properties: c. performs respiratory metabolism different from fermentative metabolism; d. does not produce toxic metabolic products or release more complex toxic substances. (9) The method of claim (1), wherein the seawater used in each tank is filtered to remove particles having a particle size of about 2.0 microns or more. (10) The method according to claim (1), wherein seawater is collected from the deep sea at least 1,000 feet below the sea surface. (11) The method according to claim (1), using a series of different antibiotics throughout the process. (12) The method according to claim (11), wherein the antibiotic is selected from the group consisting of polymyxin B, chloramphenicol, ampicillin, erythromycin, chlortetracycline, neomycin, streptomycin, and zentamycin. (13) The method according to claim (12), wherein the antibiotic selected for the particular tank is used at a concentration of about 10 parts per million in seawater. (14) The method according to claim (1), wherein the animal is a sea urchin. (15) using a series of seawater tanks to clean the adult animal, hatch the fertilized eggs, raise the hatched larvae to the point of implantation, and provide the necessary implantation surface and nutrients for further growth;
In the method of growing abalone for the first few months, a. Almost sterile seawater is used in each tank, and b. Bacterial contamination from adult animals is kept to a minimum to prevent harmful bacteria from growing in the tank. This abalone cultivation method is characterized by adding antibiotics to the seawater in each tank in order to prevent this. (16) Selected to be resistant to implantation tank antibiotics in order to promote larval implantation, suppress the increase in ammonia, and provide nutrients necessary for abalone development after implantation and metamorphosis. 16. The method of claim 15, further comprising the step of adding beneficial bacteria to the implantation tank. (17) Beneficial bacteria are ATCC (American Ty
peCultureCollection) No. 39
Claim No. 1 having the same characteristics as the characteristics of No. 451
The method described in section 6). (18) The method according to claim (16), wherein the seawater used in each tank does not contain particles having a particle size of about 0.2 μ or more at the time it is added to the tank. (19) The method according to claim (18), wherein the seawater added to each tank is filtered in advance to remove harmful bacteria and exclude particles with a particle size of about 0.2 microns or more. (20) The method of claim (18), wherein seawater is drawn from deep sea at least about 1,000 feet below sea level. (21) The method according to claim (18), wherein different antibiotics are used in each of the several tanks. (22) The method according to claim (21), wherein the antibiotic is selected from the group consisting of polymyxin B, chloramphenicol, ampicillin, erythromycin, chlortetracycline, neomycin, streptomycin, and zentamycin. (23) The method according to claim (18), which also includes the step of adding bacterial nutrients to the seawater in the implantation tank. (24) The method according to claim (23), wherein the nutrient is sodium citrate. (25) Claim No. 24, in which sodium citrate is added so that the concentration in seawater is about 2 to about 4 ppm.
The method described in section.