CN113473867A

CN113473867A - Probiotic feed for aquaculture

Info

Publication number: CN113473867A
Application number: CN201980085992.3A
Authority: CN
Inventors: K·莫克斯利; V·E·科因
Original assignee: University of Cape Town
Current assignee: University of Cape Town
Priority date: 2018-12-24
Filing date: 2019-12-24
Publication date: 2021-10-01
Also published as: KR20210106544A; WO2020136574A1; GB201821191D0; AU2019416897A1; GB2594430A

Abstract

The invention provides an aquaculture method, which comprises supplying Vibrio SY9(Vibrio midasy 9) as a food source to cultured aquatic animals. Also provided is a feed composition comprising vibrio SY 9. The aquatic animals are typically marine animals that feed on algae. These marine animals include abalone and echinoderm, such as sea urchin, sea cucumber, starfish, asterias amurensis, money of sand and lilium brownii. Vibrio SY9 may be provided on or within an alginate membrane on a support.

Description

Probiotic feed for aquaculture

Cross Reference to Related Applications

This application claims priority from uk patent application 1821191.2 filed 24.12.2018.

Technical Field

The present invention relates to a probiotic feed composition for use in aquaculture, and to a method of aquaculture comprising supplying the feed to aquaculture animals.

Background

It is known that aquaculture of marine animals such as abalone and echinoderm is very difficult. Abalone is a highly sought after seafood commodity due to its high nutritional content and unique texture, flavor and appearance organoleptic properties. Abalone is therefore a very expensive food in the international market, especially in asia. Over-fishing and hunting have reduced the number of wild abalone to the point of being threatened, and presently cultured abalone supplies the majority of abalone flesh for consumption.

While adult abalone is bred in outdoor waterways, the abalone development phase (fig. 1) from gamete fusion to the post-larval (post-larval) phase (typically about three months after colonization) occurs in indoor hatching facilities. During spawning, sexually mature adult parent fish release gametes into a separate spawning container. The free swimming sperm is then gently poured into the fertilization container, and the negatively buoyant ovum is added to the fertilization container by siphoning. After the male and female gametes are fused, the fertilized eggs sink to the bottom of the container. Fertilized eggs were collected and separated in different containers, and embryonic development occurred overnight. In this process, the embryo undergoes repeated divisions forming trochophores that swim to the top of the aqueous layer. About 24 hours after fertilization, trochophore development was complete. Thereafter, the top 70-80% of the culture water is siphoned into a larva culture container where the trochophores hatch into free-swimming planktonic larvae. Abalone will stay in the larval stage for several days (about 5 days at 20 ℃ for south african abalone (h. midae) and about 7 days at 17.5 ℃), after which they will have the ability to colonize. At this stage, the competent larvae are distributed to the prepared settling ponds at the desired settling density. In commercial hatcheries, the sedimentation surfaces are usually flat or corrugated plastic plates suspended vertically in large flow-through cells. Approximately three weeks before larvae are added, these settling surfaces are "conditioned" in the flow cell by passive seeding (i.e., incoming seawater provides colonizing microorganisms) and light. This conditioning phase promotes the growth of biofilms. Biofilms, commonly referred to as microbial communities, colonize liquid or air surface interfaces and are encapsulated in EPS (extracellular polymer) matrices. In hatchery culture, biofilms are characterized by heterogeneous populations of microalgae, particularly benthic diatoms and marine bacteria. These biofilms are the main food source for abalone in the late juvenile stage until they are weaned off to artificial feed or macroalgae at the beginning of the juvenile stage.

Unfortunately, one of the major factors limiting the optimal commercial production of abalone in south africa is the poor performance often encountered during the production incubation phase. Hatcheries are often plagued by low survival rates and low growth rates, which threaten the normal supply of premium juvenile fish during the breeding phase.

It is well known that the development of the abovementioned diatom biofilms is difficult to manage, as these biofilms tend to vary greatly in quality and quantity, and therefore they are often not sufficient to meet the energy requirements or specific nutritional requirements of the post-larval abalone. This inability to provide a consistent nutritional diet for later larval development has been recognized as a major factor in low survival and growth rates, threatening the normal supply of good quality larval fish during the abalone culture period.

Accordingly, much research has been focused on the development of artificial formulas that can provide a nutritionally complete diet for the young abalone. For example, the post-larval production system (PPS) includes alginate-based diets, including proteins, carbohydrates, and nutrient mixtures. The artificial particulate diet is coated on a rigid corrugated plastic sheet. Such diets have been reported to improve overall survival without increasing growth rate. The diet also needs to be reapplied every other day. Another artificial feed, known as the late stort's larval abalone production system (SAPPS), relies on the temperature-dependent coagulation properties of agar (0.5-1.0%). These agar formulations were mixed with artificial micro-diets and sprayed onto rigid corrugated plastic plates. The SAPPS diet improved the growth and survival of abalone in the late stages of the larvae but still required re-spraying the plates every 1-2 days. Since formulating and maintaining such feeds on abalone culture surfaces requires a lot of labor and high costs, they are not suitable for large-scale production of abalone.

Thus, while the development of artificial feed represents a promising alternative to current hatchery feeding techniques, little progress has been made in this area and there remains a need for an alternative strategy that is economically viable in a commercial environment.

Disclosure of Invention

According to a first embodiment of the present invention, there is provided a method for aquaculture of aquatic animals, the method comprising supplying at least Vibrio SY9(Vibrio midae SY9) as a food source to the aquatic animals, thereby growing the aquatic animals.

The vibrio SY9 can be provided to the aquatic animals on or in a film.

The membrane may be formed on a support.

The film may comprise a carbohydrate binder, such as alginate.

The concentration of Vibrio SY9 on or in the film may be at least 10⁸Individual cell/cm²。

The method may further comprise supplying the diatoms to the aquatic animal as an additional food source.

The diatoms may be provided on or within the same film as vibrio SY 9.

The concentration of the diatom in the film can be at least 10⁶Individual cell/cm²。

Alternatively, the method may be performed without supplying diatoms to the aquatic animal as a food source.

The method may further comprise the step of replacing the film after 5 or more days.

The aquatic animal can be a saltwater animal, such as abalone and echinoderm (e.g., sea urchin, sea cucumber, etc.).

According to a second embodiment of the invention, there is provided a feed composition formulated for feeding to aquatic animals, the feed composition comprising vibrio SY9 as a food source.

The vibrio SY9 can be coated on the film or encapsulated in the film.

The film may be formed on a flat surface.

The film may be formed from a carbohydrate binder, such as alginate.

The concentration of Vibrio SY9 in the feed composition may be at least 10⁹Individual cell/cm²。

The feed composition may also include diatoms as an additional food source for juvenile abalone.

The concentration of diatoms in the feed composition can be at least 10⁷Individual cell/cm²。

The feed composition may be stable in an aqueous composition for 5 days or more than 5 days.

Drawings

FIG. 1 shows the life cycle of abalone (Haliotis spp) (redrawn from http:// ocean link island. net/presentation/ablone/BHCAP/reduction. html).

Fig. 2 shows the survival of the later stage (post-larval h. midae) of south african abalone larvae fed with artificial biofilm consisting of either probiotic vibrio K811(v. midae K811), oval algae (coconeis sp.), or a mixture of probiotic and oval algae for 25 days, tested in three separate experiments. The initial stocking density for each tank was 150 abalones.

Detailed Description

Described herein is an aquaculture method comprising providing vibrio SY9 and a feed composition comprising vibrio SY9 as a food source to cultured aquatic animals.

Aquatic animals are generally marine animals that feed on algae, particularly animals that have a free swimming larval stage in their life cycle. The larvae settle on a suitable substrate and deform. These animals include abalone and echinoderm such as sea urchin, sea cucumber, starfish, asterias, money and lilies. In one embodiment, vibrio SY9 is provided to these animals in the post-larval stage.

Abalone is the common name for any class of marine gastropods of the family abalone (Haliotidae). Other common names are earshells (ear shells), sea ears (sea ears), mutton fish (muttonfish), mutton shells (muttonshells), ormer (worms) and baoda (p ā ua). The number of species recognized worldwide is between 30 and 130, the largest abalone in south africa being abalone in south africa (Haliotis midae). Other commercially valuable abalone species cultivated around the world include Haliotis diversicolor, black abalone in australia (Haliotis rubra), Haliotis discus hannai and red abalone (Haliotis rufescens).

The term "post-larval" describes the part of the life cycle that occurs after a swimming larva sinks from a water column to a solid, underwater surface and undergoes metamorphosis (fig. 1).

As used herein, probiotics are defined as live microbial supplements that beneficially affect a host animal by improving the intestinal balance of the host animal.

The latter young abalone moves on a solid surface through muscular feet and feeds on a biofilm of benthic diatoms and bacteria for approximately three months. Once the abalone in the later stage of the larva grows long enough (the shell is 5-10 mm long), it becomes a young abalone, ranging from feeding on benthic diatoms to withdrawal, feeding on macroalgae (seaweeds).

Researches show that the probiotics have the potential of improving the culture results of young abalones and adult abalones. However, little is known about the effect of probiotics on the early developmental stages of abalone (and aquatic species in general).

The applicant has now shown that vibrio SY9 improves the growth and survival of abalones in late larval stage in south africa without colonization of the biofilm by benthic diatoms.

Vibrio SY9 may be provided to the animal on a substrate (or surface) or on or within a membrane (or biofilm), typically formed on a support. The support may be a solid surface, such as a sheet or plate made of glass or plastics material. In one example, the support is flat, while in another example, the support is corrugated, and so forth. The solid surface may comprise a mesh to improve the adhesion of the film. Alternatively, the support may be a mesh, gauze or net-like material, which may be located within the frame. The film may be formed from a binder, such as a carbohydrate binder such as alginate, starch or agar.

Vibrio SY9 cells may be a primary or main nutrient provided to an animal. Diatoms may be optionally provided as an additional food source. Diatoms are unicellular algae with silica cell walls. Although the biofilm of benthic diatoms is produced spontaneously from influent seawater, the most common diatom species preferred for late larval abalone culture are Navicula (Navicula), amphibian (ampphirora) and oval algae (cocconiis). The method can also be carried out without providing diatoms as a food source, particularly without introducing diatoms that do not develop spontaneously in the water tank of a farm animal.

The concentration of Vibrio SY9 in the film may be at least 10⁸Individual cell/cm²And if such a concentration is present, the concentration of diatoms may be at least 10⁶Individual cell/cm²。

The film is typically replaced every 5 days to ensure a continuous supply of relatively high concentrations of probiotic bacteria. However, it is possible to try to change the film formulation so that the change period is extended, for example to 10 days or more than 10 days.

In three separate laboratory scale experiments, late larval south african abalones (average shell length 2.7 mm) were fed three different artificial biofilm feeds consisting of alginate and the putative probiotic vibrio SY9 (feed 1), a mixture of benthic diatom ootheca and vibrio SY 9K 811 (feed 2) or just ootheca (diatom control feed). The survival of abalone was monitored over 25 days and growth was quantified by measuring abalone shell length. The results confirmed that Vibrio SY9 has a probiotic effect on Haliotis diversicolor in the late stage of the larva by remarkably improving the survival rate and promoting the growth rate to be three-fold increased. These results indicate that abalones in south africa in the late larval stage can survive and grow in bacterial thin-film feeds lacking diatom cells. Therefore, the bacteria can replace diatom algae to become a main food source for the young abalone in south africa. The probiotic feed described in this study can provide a more reliable and consistent source of nutrition for the abalone in the later stage of the larvae, and has important economic significance to commercial abalone farms. Improving the later growth and survival of the larvae can greatly improve the productivity of the farm and improve the economic potential of abalone culture.

To the best of the applicant's knowledge, this is the first description of the use of probiotic microorganisms as a direct food source for abalone and sea urchins in the post-larval stage.

The invention will now be described in more detail by the following non-limiting examples, in which abalone is used as an exemplary aquaculture animal and vibrio SY9 is used as an exemplary probiotic.

Examples

Materials and methods

Microorganisms and growth media

Vibrio SY9(BCCM accession MUCL 54982) was previously grown from abalone in south Africa (Macey)&Coyne,2005) in the gastrointestinal tract. In the present study a genetically modified vibrio SY9 strain, designated K811 strain, was used to allow quantification of this bacterium in artificial feed formulations. Vibrio K811(v. midae K811) is resistant to streptomycin and contains the bioluminescent plasmid pKluxCat. This plasmid is a derivative of pAKlux2, containing the lux operon (luxCDBE) from Photorhabdus luminescens, Karsi and Lawrence 2007. Addition of the Tn9 chloramphenicol acetyltransferase (cat) gene modified pAKlux2 to enable antibiotic selection of transconjugants (unpublished data). Vibrio K811 tryptone soy broth (30g/L NaCl, 17g/L tryptone, 6g/L peptone, 2.5g/L glucose, 2.5g/L K) supplemented with 120. mu.g/ml streptomycin and 15. mu.5/ml chloramphenicol₂HPO₄pH7.8) and incubated at 150rpm and 25 ℃ in an orbital shaker.

Oval algae was selected in this study because the members of the genus are abalone larvae (long shells)>0.8 mm) biofilm feed was the primary food source. Oval algae is a single algae but not aseptically cultured algae, provided by the forestry and fisheries Department (DAFF) research aquarium (cape town, south africa). The culture was supplemented with 1ml/L metasilicic acidSodium (40g/L Na)₂SiO₃Anhydrous stock solution) and 20ml/L of Gellard (Guillard's f/2) medium (Aldrich, Sigma-Aldrich) sterile artificial seawater (24.7g/L NaCl, 4.7g/L MgCl₂·6H₂O、1.9g/L CaCl₂·2H₂O、6.3g/L MgSO₄·7H₂O、0.66g/L KCl、0.18g/L NaHCO₃pH 7.8). The culture was exposed to light at 15 ℃ for a period of 12h:12h (light intensity 4.91. mu. mol/sec/m)²) Incubate and provide ventilation for each culture through hanging air hoses.

Preparation of artificial biomembrane feed

Three alginate-based biofilm feeds were prepared: a film consisting of probiotic vibrio K811 only (feed 1), a mixed biofilm consisting of vibrio K811 and oval algae (feed 2) and a biofilm consisting of oval algae only (diatom control diet). During each 25 day growth experiment, fresh biofilms or films were prepared every 3 days.

Preparation of glass sheets

Several mosquito net meshes (11cm x 19cm) were adhered to the edges of a glass plate (11cm x 19cm,3mm thick) using marine silica gel. An Artificial Seawater (ASW) solution containing 30g/L sodium alginate (Sigma-Aldrich) was prepared and added at 0.05ml/cm²Is added to the prepared surface. The mixture was uniformly dispersed on the surface using an L-shaped glass coater. The alginate-coated surface was then dried at 25 ℃ for 24 hours and stored at-20 ℃ until needed. Preparation of Vibrio K811 cell suspension

For each new set of biofilms, cultures of vibrio K811 were grown under standard conditions for 4 hours to an OD600 of 1.0, corresponding to approximately 5x10⁸Probiotic cells/ml. The cells of each culture were obtained separately by centrifugation (10000 Xg, 10 ℃, 10 minutes). The supernatant was discarded and each bacterial cell pellet was washed twice with 100ml of ASW. Thereafter, each bacterial pellet was suspended in 10ml of ASW to reach approximately 5x10⁹Concentration of individual cells/ml. The probiotic cell suspension was used to prepare a single species biofilm consisting of vibrio K811 alone (feed 1).

Preparation of cell suspension of oval algae

For each new set of biofilms, an appropriate volume of oval algae culture was grown under standard conditions for five days up to about 10⁷Concentration of individual cells/ml. The biomass of each culture was obtained separately by centrifugation (10000 Xg, 10 ℃, 10 minutes). The supernatant was discarded and each biomass sample was washed twice with 100ml ASW. For biofilms consisting of oval algae only, diatom cell pellets were suspended in 10ml ASW to reach approximately 5x10⁷Concentration of individual cells/ml. Diatom cell suspensions were used to prepare single species biofilms consisting of oval algae only. It represents the control feed in this study.

Preparation of cell suspensions of Mixed species

Granular cells of the oval algae were suspended not in ASW but in Vibrio K811 (prepared as described above). This produced a mixed species cell suspension consisting of two microorganisms, with a final concentration of about 5x10⁹Probiotic cells/ml and about 5x10⁷Individual cells/ml of diatom. These mixed species cell suspensions were used to prepare mixed species biofilms (feed 2).

Preparation of biofilms

The respective cell suspensions were added to the prepared glass plates in a volume of 1ml per 40 square centimeters. The microorganisms were uniformly dispersed on the surface using an L-shaped glass coater. All plates were incubated at room temperature (about 25 ℃) for 20 minutes to allow the cells to attach to the alginate coated surface and then immersed in ASW to remove the less adherent cells.

Quantification of biofilm components

All biofilms were observed using an IVISx flow II imaging system (Xenogen IVIS-IIlumina II) that allowed for simultaneous detection of luminescence and/or fluorescence in vivo, and images were captured using Living Image (Living Image) software. Acquisition parameters for detecting luminescence (Vibrio K811) included a 10 second exposure, small steps, aperture 1(f/stop 1), open filter, and a 12.5cm field of view (FOV). Acquisition parameters for fluorescence detection (ootheca) included 10 second exposure, small steps, aperture 8(f/stop 8), 695-19 nm excitation filter, Cy5.5 emission filter, 12.5cm FOV. All data recorded were measured using photon flux, which represents the number of photons captured per second of exposure (p/s).

To quantify the number of microbial cells present in the biofilm at each time point, the recorded flux (p/s) data were plotted against the flux (p/s) versus microbial cells/cm²The constructed calibration curves were compared. Cells of Arthrobacter K811 were quantified using viable colony count (CFU/ml), while a counting chamber (modified Neubar, size: 0.0025 mm) was used²x 0.01mm) cells of the oval algae were quantified. It was found that the minimum number of Vibrio luminescens K811 cells required for accurate detection of luminescence and fluorescence in vivo was about 10, respectively⁴Individual cell/ml (Vibrio K811) and 10⁵Individual cells/ml (oval algae).

Verification of later-stage abalone in laboratory culture of larvae by using three artificial biofilms under standard conditions

Six glass water tanks (20 x25 cm bottom, 20 cm height, 2 mm glass thickness) were prepared to retest the water stability of three different biofilm feeds and to use standard post-larvae abalone rearing conditions (as described above). Two biofilms containing the same composition were placed horizontally on the bottom of each glass water tank where they were incubated for five days. Biofilms were removed from the tank every 24 hours, and vibrio K811 and/or the concentration of ovate remaining in the biofilms were quantified as described previously.

Growth experiment of Bao in late stage of larva

In three separate growth experiments conducted at the molecular and cell biology laboratories of the university of cape Town, south Africa, the effect of three different biofilm feeds on the growth of abalone in south Africa at the late larval stage was evaluated. The duration of each growth experiment was 25 days.

Specification of glass water tank

Six separate glass-shell water tanks (base 20x25 cm, height 20 cm, glass thickness 2 mm) were prepared and each of the three different biofilm feeds was tested in duplicate for each growth experiment. A closed recirculating water system is used. All tanks were filled to a depth of 15 cm with filtered (0.22 μm) Natural Seawater (NSW). Water change (50%) was performed by siphoning every 4-5 days to maintain good water quality. In addition, each tank was equipped with a small submersible water filter (BOYU, model SP-6011) that was run continuously throughout the growth experiment. Each tank was inflated by a hanging air hose, supplied by an air compressor (BOYU, model ACQ-007), and terminated with an air stone. The ambient temperature was maintained at 15 ℃. Light to dark photoperiods of 12h to 12h were followed. The top of each tank is covered with a black plastic cloth to limit evaporation of water and the intensity of light experienced by the abalone, which prefers low light conditions. In experiment 1, the water tank was installed directly below the overhead air conditioner. However, in experiments 2 and 3, in order to limit the evaporation of water, the water tank was placed 2 meters away from the air conditioning device, which may affect the results obtained in experiment 1.

Late stage of larva abalone in south Africa

Seafood jacobbai (jacobbabaai) (yacobubaai, south africa) donated three different batches of late juvenile south african abalones (from the same parent fish). Previously, abalone colonized and grown on natural diatom biofilms in commercial hatcheries. During hatchery collection, the animals were brushed off the plastic settlement plate surface using a large, flexible paintbrush and then transferred to a plastic container (bottom 22x26 cm, height 10 cm). This container, filled with half the sea water, was sealed and placed between the freezing bags in a refrigerator for transport to our laboratory. The time between removal of abalone from the surface of the farm and transfer to the experimental tank does not exceed 3 hours. Using a small, soft paintbrush, 150 abalones were individually transferred to each of 6 glass water tanks. At the start of each experiment, the abalones were placed individually with feet down, and their orientation was checked daily. If necessary, the abalone feet can be turned downwards by using a fur brush. Animals did not acclimate or starve before the growth test began. Mortality during the experiment was removed and quantified.

Feeding mode

Abalone is fed in a relay manner. At the start of the test, each tank with 15 cm depth of NSW was divided into two equal parts by a glass partition (20 cm x20 cm, 2 mm thick) held in a plastic clothes peg. Thereafter, a biofilm of only the probiotic strain K811 (feed 1), a mixture of two microorganisms (feed 2) or only oval algae (diatom control) was placed horizontally at the bottom half of each tank. Abalone was then added to this semi-tank, which was restricted to use for the first three days of the trial. On day 3, fresh biofilm was placed in the remaining half of each tank and the glass partition was removed, allowing abalone to move freely between the two biofilms. Animals were confined to half of the tank on the first three days of the experiment, ensuring that abalone did not get trapped in the glass bottom of the tank when the second biofilm was added to the system. On the sixth day, the first biofilm was removed and replaced with fresh biofilm. Likewise, on day 9, the second biofilm was removed and replaced. Feeding was continued in this manner, providing a layer of fresh biofilm to abalone every three days, and removing aged biofilm after six days of feeding. Cell biomass adhered to each plate was quantified immediately after biofilm preparation and six days of abalone feeding.

Quantification of abalone growth

At the start of each experiment, 50 abalone were randomly drawn from the initial herd and their shell length was measured to the nearest 0.01mm using an electronic vernier caliper. After an experimental period of 25 days, the shell length of 50 animals randomly selected from each water tank was measured. The percentage increase in the length of abalone shells fed with the different feeds and the daily growth rate (μ μ/day) were calculated.

Statistical analysis

Where applicable, data are expressed as mean and Standard Error (SE). To verify the stability in biofilm water, Student's T-test (Student's T-test) was used at T_20min,T_24hrAnd T_120hrStatistical comparisons between the two groups were performed (n ═ 4 biofilms at each time point). The effect of probiotic dietary supplements on abalone larva growth and survival was determined using one-way analysis of variance (ANOVA). To determine the effect of feed on the final average shell length (SLT25) of abalone larvae, each was considered individually in each experimentWater tank data for individual abalone as replicates (each tank n 50). For statistical analysis of% survival (and% SL increase and daily growth rate), the% survival data (or% SL increase/daily growth rate data) for all three experiments were grouped by feed (i.e., n ═ 6 different diets). At p<A statistically significant difference was established at 0.05. When the effect of ANOVA was significant, a graph-based post hoc multiple comparison method (post-hoc Tukey test) for multiple comparisons was used to examine significant differences between sample means. All analyses were performed using R version 3.0.1(R Development Core Team, 2014).

Results

The performance of three artificial feeds under the typical abalone feeding condition

T of Vibrio K811 (Table 1)_20minThere was no significant difference in concentration between bacterial biofilms (feed 1) and mixed species biofilms (feed 2) (p ═ 0.162). On average, the initial concentration of Vibrio K811 in these biofilms was 1.20X10⁸Individual cell/cm². Similarly, T of Coccomyza sp_20minThe concentration (table 1) was not significantly different (p ═ 0.107) between the diatom control feed and the mixed species biofilm (diet 2). On average, the concentration of ovate algae in these biofilms was 2.56x10⁶Individual cell/cm². The biofilm was then cultured in glass aquaria containing NSW, which has a considerable water flow due to the aeration and filtration equipment. Loss of microbial cells from different preparations was monitored over five days. There was no significant difference in the concentration of kojie algae and vibrio K811 between single and mixed species biofilms throughout the experiment.

Overall, the results of this experiment show that the concentration of each microbial component is comparable between different biofilms even after NSW extended incubation times. Although the microbial content of the biofilm was lost over the course of five days, approximately 10 days after 5 days of immersion in NSW⁵Individual probiotic cells/cm²And 5x10⁵Diatom cells/cm²Is left in the biofilm.

Growth experiment of Bao in late stage of larva

To ensure that an adequate food supply is maintained throughout the experiment, the concentration of cellular biofilm components is monitored and quantified over time. All biofilms containing Vibrio K811 or Coccomyza have a biofilm content of about 10⁸Individual probiotic cells/cm²Or 10⁶Algae cell/cm²The starting concentration of (c). Six days after abalone feeding, the concentration of each strain dropped to about 10⁵Individual probiotic cells/cm²And 10⁵Algae cell/cm²(data not shown).

Abalone fed either by biofilm of vibrio K811 alone (feed 1) or a mixture with ootheca (feed 2) showed 68% and 74% survival respectively. The control group fed oval algae alone had approximately 49% survival of abalone (table 2). Feed had a significant effect on the later survival of larvae (one-way ANOVA, F ═ 5.51, df ═ 2, p ═ 0.016). When abalone was fed with a mixture consisting of vibrio K811 and oval algae (feed 2), the survival rate was significantly higher than that of abalone fed with control feed (p ═ 0.016). When the feed consisting of probiotic alone (feed 1) was fed abalone, the survival rate was not significantly improved (p ═ 0.068) compared to the control feed. Furthermore, there was no significant difference in the survival rate of abalone fed with pure probiotic feed (feed 1) and mixed feed (feed 2) (p ═ 0.744).

Table 1: water stability of three different artificial biofilms cultured in glass water tanks equipped with aeration and water filtration devices.

Results are expressed as mean concentration (cells/cm)²) SE (n ═ 4 per biofilm).

Table 2: the number of abalones in late larval stage that survived feeding with three different artificial biofilm feeds and the corresponding percent survival (%) in three independent 25-day growth experiments. The effect of each feed was tested in duplicate and the initial feed density was 150 per water tank.

In all three experiments, the number of surviving abalones decreased during each 25-day experiment (fig. 2). However, at the end of each experiment, the survival rate was consistently higher when animals were fed probiotic feed (feed 1 and 2). In experiment 1, on day 4, a high mortality event (67 deaths) occurred in a repeat tank fed oval algae control feed. In addition, high mortality was seen on day 18 in the second repeat tank fed with the oval algae control feed (25 deaths) and in one repeat tank fed with the probiotic feed only (1, 22 deaths). This means that at the end of the experiment, there were two tanks (control tank 1 and pure probiotic tank 1) recording less than 50 shell length measurements (40 and 47 abalones respectively). No major mortality event occurred in experiments 2 and 3, and the abalone in experiment 1 was likely stressed (in the first experiment, all the glass-cased tanks were placed directly under the roof of the air conditioning unit, which could increase water evaporation and hence salinity of the water).

For experiments 1, 2 and 3, the initial mean Shell Length (SL) in the late larval stage_T0) 3.04 (+ -0.10) mm, 2.06 (+ -0.04) mm and 3.1 (+ -0.04) mm, respectively. Feed to SL of abalone in all three experiments_T25Has significant influence (one-way ANOVA, p)<0.05). In experiment 1, average SL in the late larval stage of mixed feed feeding_T25Significantly higher than the average SL in the late larval stage of the ootheca feed_T25(p<0.05). However, in experiments 2 and 3, both probiotic feeds (feed 1 and 2) significantly improved the average SL in the post-larval stage compared to the control feed_T25(p<0.05). SL from abalone fed with probiotic biofilm feed, whether the probiotic is present alone or in admixture with ootheca_T25There was no significant difference between them. (data not shown).

Shell length of abalone fed with a probiotic biofilm feed consisting of vibrio K811 alone (feed 1) or a mixture of vibrio K811 and oval algae (feed 2) increased by 21.3% and 23.0% on average, with growth rates of approximately 29.7 μm/day and 33.1 μm/day, respectively (table 3). The average shell length of abalone fed a control feed (control feed) consisting of oval algae increased only 8.3%, and the growth rate of these abalones was 11.6 μm/day. Overall, these results indicate that probiotic feed can increase the growth rate of abalones in south africa three times later in the larvae.

Table 3: in three separate 25-day growth experiments, the final average shell length (mm), percent (%) shell length increase, and daily Growth Rate (GR) (μm/day) of abalone after feeding three different artificial biofilm diets. In each experiment, each feed was tested in duplicate.

Despite the initial average Shell Length (SL) in the late stage of the larvae_T0) There were differences (experiments 1, 2 and 3 were 3.04 mm, 2.06 mm and 3.1 mm, respectively), but the percent increase in shell length and the daily growth rate data for the animals fed the probiotic feed (feeds 1 and 2) were quite consistent in all three experiments (table 3). For example, in experiments 1, 2 and 3, the average SL increase for abalone fed pure probiotic feed (feed 1) was 18.3%, 22.9% and 22.8%, respectively, while the average SL increase for abalone fed mixed feed (feed 2) was 24.9%, 22% and 22.3%, respectively. However, the results obtained with abalone fed the control feed were less consistent, with the mean SL increase for animals varying between 16.7%, 1.0% and 7.5% in experiments 1, 2 and 3, respectively. Furthermore, the average growth rates of feed-fed abalone consisting of oval algae in experiments 1, 2 and 3 were 24.4 μm/day, 0.8 μm/day and 9.6 μm/day, respectively.

Feed had a significant effect on% SL increase in abalone (one-way ANOVA, F ═ 13.69, df ═ 2, p < 0.001). The% SL increase in the post-larval stage was significantly improved (p <0.05) when abalone was fed with probiotic feed (feeds 1 and 2) compared to diatom control feed. There was no significant difference in% SL increase between abalone fed with feed consisting of vibrio K811 alone (feed 1) and abalone fed with mixed biofilm (feed 2) (p ═ 0.845). Feed also had a significant effect on growth rate of abalone (one-way ANOVA, F ═ 8.71, df ═ 2, and p ═ 0.003). The growth rate in the late phase of the larvae was significantly increased (p <0.05) when abalone was fed with probiotic feed (feed 1 and 2) compared to diatom control feed. There was no significant difference in growth rate between abalone fed with feed consisting of vibrio K811 alone (feed 1) and abalone fed with mixed biofilm consisting of vibrio K811 and oval algae (feed 2) (p ═ 0.814).

Discussion of the related Art

In the current study, three independent feeding experiments were performed to determine the effect of putative probiotic vibrio SY9 on the survival and growth of late larval abalones (Haliotis midae) when provided as the main ingredient of an artificial biofilm feed. The results show that the survival rates of the abalones fed with the probiotics (feeds 1 and 2) and the control feed in the later period of the larva are 71 percent and 49 percent respectively, and only the mixed feed (feed 2) obviously improves the survival rate of the abalones. Furthermore, in all three experiments, probiotic diet was on late SL in abalone_T25All had significant effects, with probiotic feed-fed animals having significantly higher percentage SL increase and growth rate in post-larval abalone compared to control feed-fed animals. Collectively, these results demonstrate that vibrio SY9 has a probiotic effect on abalones in south africa during the late larval stage of development.

The growth and survival results of the current study exceeded those observed in previous studies that tested the effect of alginate-based artificial feed on post-larval development (Stott et al 2002; 2003a, b). For example, in the current study, the average growth rate of late larval feeding of south african abalones with alginate probiotic feeds (feeds 1 and 2) was 29.7 μm/day and 33.1 μm/day, respectively, in three separate experiments, which was significantly higher than the average growth rate of 11.6 μm/day with control feed of diatoms in the late larval feeding. Stott et al (2003a) reported that three-day-old young black abalone (H.disc super texta) was fed with alginate feed at the later stage and supplemented with commercial feed SF-15 or SF-15[ T ] at average growth rates of 27.5 μm/day and 28.6 μm/day, respectively, for each feed. The rate of growth in the later stages of the larvae was 18.6 μm/day for the natural diatom control feed. In contrast to the current study, this study showed a higher survival rate of abalones in south africa at the late stages of larvae when fed probiotic diet (average survival rate of 71% for diets 1 and 2) compared to diatom control diet (survival rate of 49%). However, Stott et al (2003a) have reported lower post-larval survival rates for artificial diet (5.3% and 7.8% for SF-15 and SF-15[ T ], respectively) compared to diatom control diet (12.9%). This indicates that probiotic feed can provide better feeding for juvenile abalone than alginate feed described in previous studies.

Although the diatom ovate is reported to be readily digested by late larval abalone (>800 μm SL), it does not appear to be a suitable diatom for late larval south african abalones. Current studies show that there is a large difference in the growth rate in the late stages of the larvae when abalone is fed a control feed consisting of ootheca alone (table 2). Overall, these results highlight challenges associated with using diatoms as the primary food source for post-larval abalone. It would be advantageous to replace them with a formula that provides a sustained nutrient supply for later larval development.

This study showed that abalones in south africa can grow and survive later in the larvae when bacteria are the main food source. The results show that there was no significant difference in growth and survival when abalone was fed different probiotic feeds (feeds 1 and 2). This means that artificial feed based on alginate and bacterial probiotics alone may obviate the need for commercial culture of post-larval abalone with diatom biofilm.

The greatest improvement in larval later-stage survival and growth rates was observed when abalones in south africa were fed mixed biofilms (feed 2), indicating that oval algae may provide nutritional benefits not fully provided by vibrio K811 alone.

Details of microbial deposition

The vibrio SY9 strain described in this specification was deposited at the Belgian Coordinated Collections of Microorganisms (BCCM) on 1 month 7 in 2013 under the Budapest Treaty (Budapest treat) internationally recognized for the deposit of microorganisms for the purposes of patent procedures, accession number LMG P-27727.

Reference to the literature

Karsi,A.and Lawrence,M.L.,2007.Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria.Plasmid,57(3),pp.286-295.

Macey,B.M.and Coyne,V.E.,2005.Improved growth rate and disease resistance in farmed Haliotis midae through probiotic treatment.Aquaculture,245(1),pp.249-261.

Stott,A.E.,Takeuchi,T.,Koike,Y.,Yamakawa,H.and Imada,O.,2002.Using micro particle diets to replace diatoms for feeding postlarval abalone Haliotis discus discus(Reeve.).Fish.Sci.68(5),pp.1088-1093.

Stott,A.E.,Takeuchi,T.,Koike,Y.and Imada,O.,2003a.Settling and raising postlarval abalone Haliotis diversicolor supertexta(Lischke)on microparticulate diets embedded in a layer of alginate.Aquac.Res.34(7),pp.561-567.

Stott,A.E.,Takeuchi,T.,Koike,Y.,and Imada,O.,2003b.The effect of three different application methods for an artificial microparticle diet on the survival and growth rate of post-larval abalone Haliotis discus discus(Reeve).Suisanzoshoku,51(2),pp.197-203.

PCT/RO/134 Table

Claims

1. A method for aquaculture of aquatic animals, the method comprising supplying at least Vibrio SY9(Vibrio midae SY9) as a food source to the aquatic animals.

2. The method of claim 1, wherein the vibrio SY9 is supplied to the aquatic animal on or within an alginate membrane.

3. The method of claim 2, wherein the membrane is formed on a support.

4. The method of any one of claims 2 or 3, wherein the concentration of Vibrio SY9 in the membrane is at least 10⁸Individual cell/cm²。

5. The method of any one of claims 1 to 4, further comprising supplying diatoms as an additional food source to aquatic animals.

6. The method of claim 5, wherein the diatom concentration is at least 10⁶Individual cell/cm²。

7. The method of any one of claims 1 to 4, which does not include supplying diatoms as a food source to the aquatic animal.

8. The method of any one of claims 2 to 7, comprising replacing the membrane after 5 days or more of 5 days.

9. The method of any one of claims 1 to 8, wherein the aquatic animal is abalone.

10. The method of any one of claims 1 to 9, wherein the aquatic animal is a post-larval abalone.

11. The method of any one of claims 1 to 8, wherein the aquatic animal is an echinoderm.

12. A feed composition formulated for feeding to an aquatic animal, the composition comprising vibrio SY9 as a major food source.

13. The feed composition of claim 12, wherein the vibrio SY9 is coated on or encapsulated within an alginate membrane.

14. The feed composition of claim 13, wherein the membrane is formed on a support.

15. The feed composition of any one of claims 12 to 14, wherein the concentration of vibrio SY9 in the feed composition is at least 10⁹Individual cells/ml.

16. The feed composition of any one of claims 12-15, comprising diatoms as an additional food source for aquatic animals.

17. The feed composition of claim 16 wherein the diatom concentration is at least 10⁷Individual cells/ml.

18. A feed composition according to any one of claims 12 to 17 which is stable in an aqueous composition for 5 days or more than 5 days.

19. The feed composition of any one of claims 12 to 18, wherein the aquatic animal is abalone.

20. The feed composition of any one of claims 12 to 19, wherein the aquatic animal is a post-larval abalone.

21. The feed composition of any one of claims 12-18, wherein the aquatic animal is an echinoderm.