GB2594430A - Probiotic feed for post-larval abalone - Google Patents

Abstract

The invention provides a method of aquaculture comprising supplying Vibrio midae SY9 as a food source to abalone. A feed composition comprising Vibrio midae SY9 is also provided. The Vibrio midae SY9 is provided on or within an alginate film which may be formed on a support.

Description

PROBIOTIC FEED FOR POST-LARVAL ABALONE

FIELD OF THE INVENTION

The invention relates to a probiotic feed composition for post-larval abalone, and to a method of aquaculture of farmed abalone which comprises supplying the feed to post-larval abalone.

BACKGROUND TO THE INVENTION

Abalone is a highly sought-after seafood commodity due to its high nutrient content and unique sensory properties of texture, flavour and appearance. Abalone can therefore fetch high prices in international markets, especially in Asia where it is a highly-prized delicacy. Overfishing and poaching have reduced wild abalone populations to such an extent that they have become threatened, and farmed abalone now supplies most of the abalone meat consumed.

While adult abalone are cultured in outdoor raceways, the phases of abalone development (Figure 1) from gamete fusion to the late post-larval stage (usually about three months post-settlement) take place in indoor hatchery facilities. During the spawning process, sexually-mature adult broodstock release their gametes into separate spawning containers. The free-swimming sperm are then gently decanted into a fertilization container, while eggs, which are negatively buoyant, are added to the fertilization container by siphoning. After the fusion of male and female gametes, fertilized eggs sink to the bottom of the container. The eggs are collected and divided among separate containers and embryonic development occurs overnight. During this process, the embryos undergo repeated cleavage to form trochophore larvae which swim to the top layer of water. The development of trochophores is complete about 24 hours after fertilization. Thereafter, the top 70-80% of the rearing water is siphoned into larval rearing containers where the trochophores hatch into free-swimming planktonic larvae. Abalone remain in the larval phase for several days (about five days at 20°C or seven days at 17.5°C for H. midae), after which they become competent for settlement. At this stage, competent larvae are distributed to prepared settlement tanks at the desired settlement densities. In commercial hatcheries, settlement surfaces are typically flat or corrugated plastic plates which are suspended vertically in large flow-through tanks. For about three weeks prior to the addition of larvae, these settlement surfaces are "conditioned" in the flow-through tanks via passive seeding (that is, the incoming seawater provides the colonizing microorganisms) and exposure to light. This conditioning period promotes the growth of a biofilm. In hatchery culture, biofilms are characterised by a heterogeneous population of microalgae, especially benthic diatoms, and marine bacteria. These biofilms are the primary food source for post-larval abalone until they are weaned onto artificial diets or macroalgae at the start of the juvenile phase Unfortunately, one of the leading factors that limit the optimal commercial production of H. midae is the poor performance typically encountered during the hatchery phase of production.

Hatcheries are often plagued by low survival and poor growth rates which threaten the regular supply of quality juveniles to the grow-out phase of the farming process.

The development of the diatom biofilms described above is notoriously difficult to manage as these biofilms tend to be highly variable in terms of quality and quantity and, as such, they often do not adequately satisfy the energy requirements or specific nutritional needs of abalone post-larvae. This inability to provide a consistent and nutritious diet for developing post-larvae has been recognized as the major contributing factor to low survival and poor growth rates, which threaten the regular supply of quality juveniles to the grow-out phase of abalone aquaculture.

Numerous research efforts have, therefore, focused on the development of artificial formulations which can offer a nutritionally complete diet for post-larval abalone (reviewed by Takeuchi and Haga 2015). For example, Ogino and Ohta (1963) developed an alginate-based diet containing a protein, carbohydrate and nutrient mix for nutrition trials with juvenile Haliotis discus discus. This new feed system was called the Post-larval Production System (PPS) and involved painting the artificial micro-particulate diet onto hard corrugated plastic plates, using 2% sodium alginate as a binder. Stott et al. (2002) modified the alginate method used in the PPS by dipping the painted alginate plates into a solution of 5% calcium chloride. This cross-linking agent converts alginate into an insoluble gel which improves the water stability of the feed. These diets improved overall survival but did not improve growth. The diets also needed to be re-coated every second day. Stott et al. (2004) developed an alternative artificial diet, called Stott's Abalone Postlarval Production System (SAPPS), which relies on the temperature-related setting properties of agar (0.5-1.0%). These agar formulations are mixed with an artificial micro-diet and sprayed onto hard corrugated plastic plates. The SAPPS diet improved the growth and survival of post-larval abalone, but the plates still needed to be re-sprayed every 1-2 days. Due to the intensive labour and high costs required for formulating and maintaining all of the above diets on abalone culturing surfaces, they are not suitable for large-scale abalone production.

Thus, while the development of artificial feeds represents a promising alternative to current hatchery feeding technologies, Takeuchi and Haga (2015) confirm that little progress has been made in the field, and there remains a need for an alternative strategy which would be economically-viable in a commercial situation.

SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided a method of aquaculture of farmed abalone, the method including supplying at least Vibrio midae SY9 as a food source to post-larval abalone, thereby allowing the post-larval abalone to grow.

The Vibrio midae SY9 may be supplied to the post-larval abalone on or within a biofilm.

The biofilm may be formed on a support.

The biofilm may include a carbohydrate binder, such as alginate.

The concentration of the Vibrio midae SY9 in the biofilm may be at least 108 cells/cm2.

The method may further include supplying diatoms as an additional food source to the post-larval abalone.

The diatoms may be supplied on or within the same biofilm as the Vibrio midae SY9.

The concentration of the diatoms in the biofilm may be at least 106 cells/cm2.

Alternatively, the method may be performed without supplying diatoms as a food source to the post-larval abalone.

The method may also include the step of replacing the biofilm after 5 or more days.

According to a second embodiment of the invention, there is provided a feed composition formulated for feeding post-larval abalone, the feed composition including Vibrio midae SY9 as a food source.

The Vibrio midae SY9 may be coated onto or encapsulated within a biofilm.

The biofilm may be formed on a solid surface.

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

The concentration of Vibrio midae SY9 in the feed composition may be at least 109 cells/mi.

The feed composition may also include diatoms as an additional food source for the post-larval abalone.

The concentration of the diatoms in the feed composition may be at least 107 cells/ml.

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

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the life history of the abalone, Hattori& spp. (redrawn from: http://oceanlink island.net/Conservation/abalone/BHCAP/development.html).

Figure 2 shows survival of post-larval H. midae fed artificial biofilm diets consisting of either probiotic V midae K811, Cocconeis sp., or a mixture of both probiofic and Cocconeis sp. for 25 days, tested in three separate experiments. Each tank had an initial stocking density of 150 abalone.

DETAILED DESCRIPTION OF THE INVENTION

A method of aquaculture of farmed abalone comprising supplying Vibrio midae SY9 as a food source to post-larval abalone is described herein, together with a feed composition for post-larval abalone comprising Vibrio midae SY9.

Abalone is a common name for any of a group of marine gastropod molluscs in the family Haliotidae. Other common names are ear shells, sea ears, muttonfish, muttonshells, ormer and paua. The number of species recognized worldwide ranges between 30 and 130. The largest abalone in South Africa is Haliotis midae. Other commercially important abalone species farmed in various regions around the world include Haliotis divers/color, Haliotis rubra, Haliotis discus hannai and Haliotis rufescens.

As used herein, a biofilm is defined as a surface-attached community of microorganisms which forms three-dimensional colonies enmeshed in a polysaccharide film.

The term "post-larval" describes the portion of the abalone life cycle that occurs after the swimming veliger larvae settle from the water column onto solid, submerged surfaces and undergo metamorphosis (Figure 1). The post-larval abalone are motile on solid surfaces by way of a muscular foot and feed on a biofilm of benthic diatoms and bacteria for a period of approximately three months. Once the post-larval abalone has grown sufficiently (5 -10 mm shell length), it becomes a juvenile abalone that is weaned from a diet of benthic diatoms to feeding on macroalgae (seaweed).

As used herein, a probiotic is defined as live microbial supplement which beneficially affects the host animal by improving its intestinal balance.

Studies have demonstrated the potential for probiotics to improve outcomes of juvenile and adult abalone culture. For example, following the first suggestion by Erasmus et al. (1997) that the enteric bacteria of the South African abalone could enhance the growth rate of the host, Macey and Coyne (2005) demonstrated that the authochthonous bacterium Vibrio midae SY9 enhances the nutrition and health of small (20 mm) and large (67mm) Haliotis midae. Hadi et al. (2014) also showed that probiofic-supplemented feeds significantly improved the growth and reduced mortality of juvenile H. iris (20-30 mm). While these studies have demonstrated the potential for probiotics to improve outcomes of juvenile and adult abalone culture, very little is known about the influence of probiotics on abalone (and aquatic species in general) during their early developmental phases (reviewed by Vine et al. 2006).

As mentioned above, the autochthonous bacterium Vibrio midae SY9 has been shown to enhance the nutrition and health of juvenile South African Haliotis midae, but the effect of this bacterium on post-larval H. midae has not previously been explored. The applicant has now shown that V. midae SY9 can improve the growth and survival of post-larval H. midae in the absence of benthic diatoms colonising the biofilm.

Vibrio midae SY9 can be supplied to the post-larval abalone on or within a biofilm, typically formed on a support. The support can be a solid surface, such as a sheet or plate made from glass or a plastics material. In one example, the support is flat and in another example the support is corrugated or the like. The solid surface may contain a mesh-type fabric to improve adherence of the biofilm. Alternatively, the support can be a net, gauze or mesh-like material, which can be located within a frame. The biofilm can be formed from a binder, for example a carbohydrate binder such as alginate, starch or agar.

Diatoms can optionally be provided as an additional food source to the post-larval abalone, although the method can be performed without supplying diatoms as a food source, particularly without introducing diatoms that haven't developed spontaneously in the tank in which the abalone are kept. Diatoms are single-celled algae which have a cell wall of silica. Although biofilms of benthic diatoms are generated spontaneously from the inflowing seawater, common diatoms preferred for post-larval abalone culture include Navicula, Amphiprora and Cocconeis species.

The concentration of the Vibrio midae SY9 in the biofilm may be at least 108 cells/cm2, and if present, the concentration of the diatoms may be at least 108 cells/cm2.

The biofilm is typically replaced every 5 days to ensure a constant supply of probiotic at relatively high concentrations. However, it is envisaged that varying the formulation of the biofilm could result in the extension of the replacement period, such as to 10 or more days.

In three separate laboratory-scale experiments, post-larval H. midae (mean shell length 2.7 mm) were fed three different artificial biofilm diets which consisted of alginate and either putative probiotic V. midae SY9 only (diet 1), a mixture of the benthic diatom Cocconeis sp. and Vibrio midae SY9 K811 (diet 2) or Cocconeis sp. only (diatom control diet). Abalone survival was monitored over 25 days, and growth was quantified by measuring abalone shell length. Results confirmed that V. midae SY9 has a probiotic effect on post-larval H. midae by significantly improving survival and promoting a three-fold increase in the growth rate of these animals. Results also demonstrated that post-larval H. midae can survive and grow on a bacterial biofilm diet lacking diatom cells, suggesting that bacteria could replace diatoms as the principle food source for post-larval H. midae. The probiotic diets described in this study could have important economic implications for commercial abalone hatcheries by offering a more reliable and consistent source of nutrition for abalone post-larvae. Improving post-larval growth and survival could greatly enhance hatchery productivity and boost the economic potential of abalone aquaculture.

To the applicant's knowledge, this is the first time that the use of probiotic microorganisms as a direct food source for post-larval abalone has been described.

The invention will now be described in more detail by way of the following non-limiting examples.

Examples

Materials and methods Microorganisms and growth media Previously, Macey (2005) isolated Vibrio midae SY9 (BCCM Accession No. MUCL 54982) from the digestive tract of adult H. midae. The use of a genetically-modified strain of Vibrio midae SY9, designated strain K811, was used in this study to allow for the quantification of this bacterium in artificial feed formulations. V. midae K811 is resistant to streptomycin and contains the bioluminescent plasmid pKluxCat. This plasmid is a derivative of pAKlux2 containing the lux operon (luxCDABE) from Photorhabdus luminescens (Karsi and Lawrence 2007). pAKlux2 was modified by the addition of the Tn9 chloramphenicol acetyltransferase (cat) gene to enable antibiotic selection of transconjugants (unpublished data). V midae K811 was routinely grown in tryptone soy broth (30 g/L NaCI, 17 g/L tryptone, 6 g/L peptone, 2.5 g/I glucose, 2.5 g/L K2HPO4, pH7.8) supplemented with 120 pg/ml streptomycin and15 pg/ml chloramphenicol and incubated in an orbital shaker at 150 rpm and 25°C.

Cocconeis sp. was chosen for this study because members of this genus are the principle food source for abalone post-larvae (>0.8 mm in shell length) feeding on biofilms (Kawamura and Takami, 1995). A unialgal but non-axenic culture of Cocconeis sp. was supplied by the Department of Forestry and Fisheries (DAFF) Research Aquarium (Cape Town, South Africa). Cultures were routinely grown in sterile artificial seawater (24.7 g/LNaCI, 4.7 g/L MgC12*6 H20, 1.9 g/L CaC12*2H20, 6.3 g/L MgSO4-7H20, 0.66 g/LKCI, 0.18 g/L NaHCO3, pH 7.8) supplemented with 1 ml/L sodium metasilicate (40 g/L Na2SiO3anhydrous stock solution) and 20mI/L Guillard's f/2 medium (Sigma-Aldrich). Cultures were incubated at 15°C under a 12h:12h light:dark photoperiod (light intensity 4.91 pmol/sec/m2) and aeration was supplied by suspending an air hose in each culture.

Artificial biofilm diet preparation Three alginate-based biofilm diets were prepared: biofilms consisting of only probiotic V. midaeK811 (diet 1), mixture biofilms consisting of both V. midaeK811 and Cocconeis sp.

(diet 2), and biofilms consisting of only Cocconeis sp. (diatom control diet). Fresh biofilms were prepared every three days over the course of each 25-day growth experiment.

Preparation of glass plates Pieces of mosquito netting (11 cm x 19 cm) were adhered to the edges of glass plates (11 cm x 19 cm, 3 mm thickness) using marine silicone. A solution containing 30 g/L sodium alginate (Sigma-Aldrich) was prepared in artificial seawater (ASW) and was added to the prepared surfaces at a volume of 0.05 ml/cm2. The mixture was evenly dispersed over the surfaces using an L-shaped glass spreader. The alginate-coated surfaces were then dried for 24 hours at 25°C before being stored at -20°C until needed.

Preparation of Vibrio midae K811 cell suspension For each new set of biofilms, cultures of Vibrio midae K811 were grown under standard conditions for four hours to 0D600=1.0, equivalent to approximately 5 x 108 probiotic cells/ml.

The cells from each culture were harvested separately by centrifugation (10 000xg, 10°C, 10 mins). The supernatant was discarded and each bacterial cell pellet was washed twice with 100 ml of ASW. Thereafter, each bacterial pellet was suspended in 10 ml of ASW to achieve a concentration of approximately 5 x 109 cells/ml. Probiotic cell suspensions were used to prepare single-species biofilms consisting of only V. midae K811 (diet 1).

Preparation of Cocconeis sp. cell suspension For each new set of biofilms, appropriate volumes of Cocconeis sp. cultures were grown under standard conditions for five days to a concentration of approximately 107 cells/ml. The biomass from each culture was harvested separately by centrifugation (10 000xg, 10°C, 10 mins). The supernatant was discarded and each biomass sample was washed twice with 100 ml of ASW. For biofilms consisting of only Cocconeis sp., the diatom cell pellets were suspended in 10 ml of ASW to achieve a concentration of approximately 5 x 107 cells/ml. Diatom cell suspensions were used to prepare single-species biofilms consisting of only Cocconeis sp. which represented the control diet in this study.

Preparation of mixed-species cell suspension Pelleted cells of Cocconeis sp. were not suspended in ASW but rather in suspensions of V. midae K811 (prepared as described above). This produced a mixed-species cell suspension consisting of both microorganisms at a final concentration of approximately 5 x 109 probiofic cells/ml and approximately 5 x 107 diatom cells/ml. These mixed-species cell suspensions were used to create mixed-species biofilms (diet 2).

Preparation of biofilms The respective cell suspension was added to prepared glass plates at a volume of 1 ml per 40 cm2. The microorganisms were dispersed evenly over the surfaces using an L-shaped glass spreader. All plates were incubated at room temperature (approximately 25°C) for 20 minutes to allow the cells to attach to the alginate-coated surfaces, and then dipped in ASW to remove weakly-adhering cells.

Quantification of biofilm components All biofilms were viewed using the Xenogen IVIS-Illumina II, which allows for simultaneous detection of luminescence and/or fluorescence in vivo, and images were captured using Living Image software. Acquisition parameters for the detection of luminescence (V. midae K811) included 10 sec exposure, small binning, f/stop 1, open filter and 12.5 cm field-of-view (FOV).

Acquisition parameters for the detection of fluorescence (Cocconeis sp.) included 10 sec exposure, small binning, f/stop 8, 695-770 nm excitation filter, Cy5.5 emission filter, 12.5 cm FOV. All data recorded were measured using photon flux, which represents the number of photons of light captured per second of exposure (p/s).

To quantify the number of microbial cells present in the biofilms at each time point, the recorded flux (p/s) data were compared to established calibration curves which were constructed by plotting flux (p/s) versus microbial cells/cm2. Cells of V. midae K811 were quantified using viable plate counts (CFU/ml), while cells of Cocconeis sp. were quantified using a counting chamber (Neubauer improved, dimensions: 0.0025 mm2 x 0.01 mm). The minimum number of luminescent V. midae K811 cells required for accurate detection of luminescence and fluorescence in vivo was found to be approximately 104 cells/cm2 (V. midae K811) and 105 cells/cm2 (Cocconeis sp.), respectively.

Validation of three artificial biofilms under typical conditions used for laboratory-based culture of post-larval abalone Six glass tanks (base 20 x 25 cm, height 20 cm, glass thickness 2 mm) were prepared to test the water stability of three different biofilm diets in duplicate and using typical housing conditions for post-larval abalone (described above). Two biofilms containing the same components were placed horizontally at the bottom of each glass tank where they were incubated for a total of five days. The biofilms were removed from the tanks every 24 hours and the concentration of V. midae K811 and/or Cocconeis sp. remaining in the biofilms was quantified as described previously.

Post-larval H. midae cirowth experiments The effect of the three different biofilm diets on the growth of post-larval H. midae was assessed in three separate growth experiments conducted in the Molecular and Cell Biology laboratories at the University of Cape Town, South Africa. The duration of each growth experiment was 25 days.

Specifications for glass housing tanks

For each growth experiment, six separate glass housing tanks (base 20 x 25 cm, height 20 cm, glass thickness 2 mm) were prepared to test each of the three different biofilm diets in duplicate. A closed, re-circulating water system was used. All tanks were filled to a depth of 15 cm with filtered (0.22 pm) natural seawater (NSW). Water changes (50%) were performed by siphoning every 4-5 days to maintain good water quality. In addition, each tank was fitted with a small submersible water filter (BOYU, model SP-6011) which ran continuously throughout the growth experiment. Each tank was aerated by suspending an air hose, fed by an air compressor (BOYU, model ACQ-007) and terminating with an air stone, in each tank. The ambient temperature was kept at 15°C. A photoperiod of 12h:12h light:dark was followed. The top of each tank was covered with black plastic sheets to limit water evaporation as well the light intensity experienced by the abalone, which prefer low light conditions. During experiment 1, the tanks were housed directly below an overhead air-conditioning unit.

However, for experiments 2 and 3, the tanks were housed 2 meters away from the air-conditioning unit in order to limit water evaporation which may have impacted the results obtained in experiment 1.

Post-larval H. midae Three separate batches of post-larval H. midae (originating from the same broodstock) were donated by Jacobsbaai Sea Products (Jacobsbaai, South Africa). Previously, the abalone had been settled and grown on natural diatom biofilms within the commercial hatchery facility.

During collection from the hatchery, the animals were brushed off the surface of the plastic settlement plates using a large, soft paintbrush and transferred to a plastic container (base 22 x 26 cm, height 10 cm). The container was half-filled with seawater, tightly sealed and placed between frozen ice-packs in a cooler box for transportation to our laboratories. The time between removing the abalone from farm surfaces and transferring them to the experimental tanks did not exceed 3 hours. Using a small, soft paint brush, 150 abalone were individually transferred to each of 6 glass tanks. The abalone were individually placed foot-down at the start of each experiment and their orientation was checked daily. If necessary, abalone were turned foot-down using a soft paintbrush. The animals were not acclimatized or starved before the commencement of the growth trial. Mortalities during the experimental period were removed and quantified.

Feeding regime The abalone were fed according to a relay-type regime. At the start of the trial, each tank containing NSW to a depth of 15 cm was divided into two equal halves using a glass divider (20 cm x 20 cm, 2 mm thickness) held in place with plastic clothes pegs. Thereafter, one biofilm containing either probiotic strain K811 only (diet 1), a mixture of both microorganisms (diet 2) or Cocconeis sp. only (diatom control) was placed horizontally at the bottom of one half of each tank. Abalone were then added to this half of the tank where they were restricted for the first three days of the trial. On day three, a fresh biofilm was placed in the remaining half of each tank and the glass divider was removed, thereby permitting the abalone to move freely between the two biofilms. Restricting the animals to one half of the tank for the first three days of the trial ensured that the abalone would not be trapped on the glass bottom of the tank when the second biofilm was added to the system. On day six, the first biofilm was removed and replaced with a fresh biofilm. Similarly, on day nine, the second biofilm was removed and replaced. Feeding continued in this manner whereby one fresh biofilm was presented to the abalone every three days, and ageing biofilms were removed after being grazed for six days. The cellular biomass adhering to each plate was quantified immediately after biofilm preparation as well as after six days of abalone grazing.

Quantification of abalone growth At the start of each experiment, 50 abalone were randomly selected from the initial pool of animals and the shell lengths were measured to the nearest 0.01 mm using electronic Vernier callipers. After the 25-day experimental period, the shell lengths of 50 randomly-selected animals from each tank were measured. The percentage increase in shell length, as well as the daily growth rate (pm/day), of abalone fed different diets were calculated.

Statistical analyses Data is presented as means and standard error (SE) where applicable. For the validation of biofilm water stability, Student's T-test was used for statistical comparison between two groups at T2Omin, T24h1 and T120h1 (n = 4 biofilms at each time point). The effect of probiofic dietary supplementation on the growth and survival of post-larval abalone was determined using one-way analysis of variance (ANOVA).To determine the effect of diet on the final mean shell length (5L125) of post-larval abalone, data for each tank was considered separately in each experiment so that individual abalone served as replicates (n=50 for each tank). For the statistical analysis of % survival (as well as of %SL increase and daily growth rate), the % survival data (or %SL increase/daily growth rate data) for all three experiments were grouped by diet (that is, n=6 for each of three different diets). Statistically significant differences were established at p<0.05.VVhen the effects of ANOVA were significant, the post-hoc Tukey test for multiple comparisons was used to test for 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 diets under typical abalone housing conditions The T2ornin concentration of V. midae K811 (Table 1) was not significantly different (p = 0.162) between bacterial biofilms (diet 1) and mixed-species biofilms (diet 2). On average, the initial concentration of V. midae K811 in these biofilms was 1.20 x 108 cells/cm2. Similarly, the Tzomin concentration of Cocconeis sp. (Table 1) was not significantly different (p = 0.107) between the diatom control diet and mixed-species biofilms (diet 2). On average, the initial concentration of Cocconeis sp. in these biofilms was 2.56 x 106 cells/cm2. The biofilms were subsequently incubated in glass aquaria containing NSW that had a considerable water flow due to the aeration and filtration apparatus. The loss of microbial cells from the different preparations was monitored over five days. There were no significant differences in the concentration of Cocconeis sp. and V midae K811 between single-and mixed-species biofilms throughout the experiment.

Table 1: The water stability of three different artificial biofilms incubated in glass aquaria fitted with aeration and water filtration units. Results are expressed as mean concentration (cells/cm2) ±SE (n=4 for each biofilm).

Biofilm diet Cells/cm2(±SE) 20min 24 hrs 48 his 72 his 96 his 120 hrs Single-Species Control: Cocconeis sp 2.42E+06 1.32E+06 1.11E+06 9.10E+05 8.55E+05 5.67E+05 (±1.34E+05) (±1.30E+05) (±1.24E+05) (±1.37E+05) (±8.52E+04) (±4.38E+04) Diet 1:V. midae K811 1.17E+08 5.06E+07 9.63E+06 5.04E+06 1.29E+06 1.61E+05 (±4.50E+06) (±1.15E+07) (±4.13E+06) (±2.56E+06) (±7.70E+05) (±7.33E+04) Diet 2: Mixed-Species V. midae K811 component 1.23E+08 4.92E+07 2.64E+06 1.67E+06 2.68E+05 8.31E+04 (±4.23E+06) (±2.09E+06) (±2.57E+05) (±4.48E+05) (±3.50E+04) (±1.44E+04) Cocconeis sp component 2.71E+06 1.04E+06 8.07E+05 7.79E+05 7.35E+05 6.85E+05 (±1.62E+05) (±1.67E+05) (±8.37E+04) (±6.62E+04) (±7.07E+04) (±6.27E+04) Overall, results from this experiment demonstrate that the concentration of each microbial component is comparable between different biofilms, even after extended incubation in NSW.

Despite the loss of microbial components from the biofilms over the course of five days, approximately 105 probiotic cells/cm2 and 5 x 105 diatom cells/cm2 remained in the biofilms following their immersion for 5 days in NSW.

Post-larval H. midae qrowth experiments In order to ensure that an adequate food supply was maintained throughout the trial, the concentration of the cellular biofilm components were monitored and quantified over time. All biofilms containing V. midae K811 or Cocconeis sp. had a starting concentration of approximately 108 probiotic cells/cm2 or 106 algal cells/cm2, respectively. After six days of abalone grazing, the concentration of each strain decreased to approximately 105 probiotic cells/cm2 and 106 diatom cells/cm2 (data not shown).

Abalone fed biofilms consisting of V. midae K811either alone (diet 1) or in a mixture with Cocconeis sp. (diet 2) displayed survival rates of 68% and 74%, respectively. The survival rate of abalone fed a control diet consisting of Cocconeis sp. was about 49% (Table 2). Diet had a significant effect on post-larval survival (One-Way ANOVA, F=5.51, df=2, p= 0.016).

Survival was significantly greater when abalone were fed a diet consisting of a mixture of V. midaeK811 and Cocconeis sp. (diet 2) than when abalone were fed a control diet (p= 0.016). Survival was not significantly improved (p=0.068) when abalone were fed a diet consisting of probiotic only (diet 1) compared to a control diet. In addition, there was no significant difference (p=0.744) between survival of abalone fed a probiotic only diet (diet 1) and a mixture diet (diet 2).

Table 2: The number of surviving post-larval H. midae, and the corresponding percentage (%) survival, following abalone fed on three different artificial biofilm diets in three separate 25-day growth experiments. The effects of each diet were tested in duplicate and the initial stocking density was 150 animals/tank.

Biofilm diet Experiment 1 Experiment 2 Experiment 3 Mean Mean % No. surviva abalon I e (±SE) No. % No. % No. ok abalon surviva abalon surviva abalon surviva e I e I e I Control diet Cocconeis sp. 40 27 93 62 72 48 68 (±11) 49 (1) Cocconeis sp. 67 45 81 54 84 56 77 (±4) (2) Diet 1 V. midae K811 47 31 117 78 123 82 95(±17) 68 (1) V. midae K811 90 60 107 71 125 83 107(±7) (2) Diet 2 Mixture (1) 111 74 103 69 109 73 107(±2) 74 Mixture (2) 107 71 113 75 118 79 112(±2) In all three experiments, the number of surviving abalone declined throughout each 25-day experimental period (Figure 2). However, by the end of each experiment, survival was consistently higher when the animals were fed a probiotic diet (diets 1 and 2). In experiment 1, there was a high mortality event (67 deaths) at day 4 in one replicate tank containing animals fed a Cocconeis sp. control diet. In addition, high mortalities were also seen at day 18 in the second replicate tank containing animals fed a Cocconeis sp. control diet (25 deaths), as well as in one replicate tank containing animals fed a probiotic-only diet (diet 1, 22 deaths). This meant that at the end of the experiment, there were two tanks (control tank 1 and probiotic-only tank 1) where less than 50 shell length measurements (40 and 47 abalone, respectively) were recorded. There were no large mortality events in experiments 2 and 3, and it is possible that the abalone in experiment 1 may have been stressed On the first experiment, all glass housing tanks were maintained directly under an overhead air-conditioning vent which could have increased water evaporation and, in turn, water salinity).

The initial mean shell length (SL-m) of post-larvae was 3.04 (±0.10) mm, 2.06 (±0.04) mm and 3.1 (±0.04) mm for experiments 1, 2 and 3, respectively. Diet had a significant effect on the SLT25 of abalone in all three experiments (One-Way ANOVA, p <0.05). In experiment 1, the mean SLT25 of post-larvae fed a mixture diet was significantly greater than the mean SLT25 of post-larvae fed the Cocconeis sp. diet (p <0.05). However, in experiments 2 and 3, both probiotic diets (diets 1 and 2) significantly improved the mean SLT25 of post-larvae compared to the control diet (p <0.05). There was no significant difference between the SLT25 of abalone fed probiotic biofilm diets, irrespective of whether the probiotic was present alone or as a mixture with Cocconeis sp. (data not shown).

Abalone fed a probiotic biofilm diet consisting of V. midae K811 only (diet 1) or a mixture of V. midae K811 and Cocconeis sp. (diet 2) exhibited, on average, a 21.3% and 23.0% improvement in shell length with a growth rate of about 29.7 pm/day and 33.1 pm/day, respectively (Table 3). The average shell length of abalone fed a control diet consisting of Cocconeis sp. (control diet) improved by only 8.3%, and the growth rate of these abalone was 11.6 pm/day. Overall, these results indicate that a probiotic diet can triple the growth rate of post-larval H. midae.

Table 3: Final mean shell length (mm), percentage (%) shell length increase and daily growth rate (GR) (pm/day) of abalone after being fed three different artificial biofilm diets in three separate 25-day growth experiments. Each diet was tested in duplicate in each experiment.

Biofilm diet Mean Mean GR Experiment 1 Experiment 2 Experiment 3 % SL (pm/d) Inc. (± SE) (± SE) Mean S LT25 (±SE) mm % GR Mean SL T25 (±SE) mm % GR Mean SL T25 (±SE) mm % GR SL (pm/ SL (pm/d) SL (pm/d inc. d) inc. inc.

Control diet Cocconeis sp. 3.73 18.5 27.6 2.08 1.1 0.8 3.25 4.7 6.0 8.3 11.6 (1) (±0.14) (±0.05) (±0.09) (±2.99) (±4.53) Cocconeis sp. 3.57 14.8 21.2 2.08 0.9 0.8 3.43 9.6 13.2 (2) (±0.10) (±0.04) (±0.08) Diet 1 V. midaeK811 3.55 14.4 20.4 2.86 27. 32.0 3.94 21.3 33.6 21.3 29.7 (1) (±0.15) (±0.06) 9 (±0.12) (±1.94) (±3.50) V. midaeK811 3.91 22.2 34.7 2.51 17. 18.0 4.09 24.3 39.6 (2) (±0.13) (±0.07) 8 (±0.11) Diet 2 Mixture (1) 3.97 23.4 37.2 2.76 25. 28.0 4.09 24.2 39.6 23.0 33.1 (±0.15) (±0.07) 5 (±0.10) (±1.26) (±3.66) Mixture (2) 4.13 26.4 43.6 2.53 18. 18.8 3.89 20.3 31.6 (±0.11) (±0.07) 4 (±0.13) Despite differences in the initial mean shell length (SL-r0) of post-larvae (3.04 mm, 2.06 mm and 3.1 mm for experiments 1, 2 and 3, respectively), the % shell length increase and daily growth rate data for the animals fed probiotic diets (diets 1 and 2) were fairly consistent between all three experiments (Table 3). For example, the mean SL increase of abalone fed a probiotic only diet (diet 1) was 18.3%, 22.9% and 22.8% in experiments 1, 2 and 3, respectively, while that of abalone fed a mixture diet (diet 2) was 24.9%, 22% and 22.3%, respectively. However, there was poor consistency in the results obtained for the animals fed the control diet where the mean SL increase of the animals varied between 16.7%, 1.0% and 7.5% in experiments 1, 2 and 3, respectively. In addition, the mean growth rate of abalone fed a diet consisting of Cocconeis sp. was 24.4 pm/day, 0.8 pm/day and 9.6 pm/day in experiments 1, 2 and 3, respectively.

Diet had a significant effect on the % SL increase of abalone (One-Way ANOVA, F=13.69, df=2, p <0.001). The %SL increase of post-larvae was significantly improved when abalone were fed a probiotic diet (diet 1 and 2) compared to a diatom control diet (p <0.05). There was no significant difference (p = 0.845) in % SL increase between abalone fed a diet consisting of V midae K811 only (diet 1), and abalone fed a mixture biofilm (diet 2). Diet also had a significant effect on abalone growth rate (One-Way ANOVA, F=8.71, df = 2, p=0.003). The growth rate of post-larvae was significantly improved (p <0.05) when abalone were fed a probiotic diet (diets 1 and 2) compared to a diatom control diet. There was no significant difference (p= 0.814) in growth rate between abalone fed a diet consisting of V. midae K811 only (diet 1), and abalone fed a mixture biofilm consisting of V midae K811 and Cocconeis sp. (diet 2).

Discussion In the current study, three independent feeding experiments were conducted to determine the impact of the putative probiotic Vibrio midae SY9 on the survival and growth of post-larval South African abalone, Haliotis midae, when provided as a principle component of an artificial biofilm diet. Results demonstrated that the survival of post-larval H. midae fed probiotic (diets 1 and 2) and control diets was 71% and 49%, respectively, but only a mixture diet (diet 2) significantly improved abalone survival. In addition, probiotic diets had a significant effect on the SLT25 of post-larvae in all three experiments, and the %SL increase and growth rates of post-larval abalone fed a probiotic diet were significantly higher compared to animals fed the control diet. Overall, these results confirm that V midae SY9 has a probiotic effect on H. midae during the post-larval phase of development.

The growth and survival outcomes of the current study match or surpass those observed in previous studies which tested the effects of alginate-based artificial diets on post-larval performance (Stott et al. 2002; 2003a,b). For example, in the current study, the mean growth rate of H. midae post-larvae fed alginate-based probiotic diets (diets 1 and 2) in three separate experiments was 29.7 pm/day and 33.1 pm/day, respectively, which was significantly higher than the mean growth rate of 11.6 pm/day observed for post-larvae fed a diatom control diet.

Stott et al. (2003a) fed three-day-old H. discus supertexta post-larvae with alginate-based diets supplemented with the commercial diets SF-15 or SF-15[T] and reported mean growth rates of 27.5 pm/day and 28.6 pm/day, for each diet respectively. The growth rate of post-larvae fed a natural diatom control diet was 18.6 pm/day. However, in contrast to the current study which demonstrated that the survival of post-larval H. midae was better when they were fed a probiotic diet (mean survival of 71% for diets 1 and 2) compared to a diatom control (49% survival), Stott et al. (2003a) reported lower post-larval survival rates on the artificial diets (5.3% and 7.8% on SF-15 and SF-15[T], respectively) compared to the diatom control (12.9%). This suggests that probiotic formulations could provide a superior diet for post-larval abalone compared to the alginate formulations described in other studies.

Cocconeis spp. do not appear to be a suitable diatom for post-larval H. midae, even though this diatom is reported to be highly digestible by post-larval abalone (>800 pm SL) (Takami, 2003; Kawamura et al. 1998; Onitsuka et al. 2007). The current study showed a wide variability in post-larval growth rates when abalone were fed a control diet consisting of only Cocconeis sp. (Table 2). Overall, these results highlight the challenges associated with the use of diatoms as a principle food source for post-larval abalone. It would be advantageous to replace them with formulated diets that provide a consistent supply of nutrients for developing post-larvae.

Interestingly, this study has demonstrated that post-larval H. midae can grow and survive when bacteria are supplied as the principle food source. Results showed that the growth and survival rates were not significantly different when abalone were fed different probiotic diets (diets 1 and 2). This suggests that artificial diets based solely on alginate and bacterial probiotics may preclude the need for diatom biofilms for the commercial rearing of post-larval abalone.

Results of the current study show that the greatest improvement in post-larval survival and growth rate was observed when post-larval H. midae were fed a mixed biofilm (diet 2) which suggests that Cocconeis sp. may offer nutritional benefits which V midae K811 alone cannot fully provide.

Results of this study showed that the concentration of V. midae K811 in the artificial diets presented to abalone was approximately 108 probiotic cells/cm2. After 6 days of grazing, the concentration of probiotic decreased to 106 probiotic cells/cm2. According to Britz (1996), the feed consumption rate of adult H. midae (SL 21.2 ± 2.5 mm) fed formulated diets ranged from 0.5-0.8% of their body weight per day. Considering that an abalone measuring 2 mm in SL weighs only about 10 mg (data not shown), it is unlikely that the large loss of probiotic from surfaces is due to direct feeding. Rather, this loss is probably due to a high leaching rate of the probiotic from the alginate surfaces, as demonstrated in the biofilm water stability experiment. This can be overcome by using water-stable formulations of the probiotic.

In the current study, "stability" of the artificial biofilms was measured in terms of V. midae K811 leaching which, in turn, was assayed by monitoring bacterial luminescence in biofilms. It is important to note that bioluminescence is an enzymatic reaction and that, as a result, the amount of luminescence produced per cell is not a fixed value. Therefore, the main challenge associated with monitoring V. midae K811 based on expression of this phenotype is that the ability of luminescent bacterial cells to emit light is dependent on the metabolic activity of cells which, in turn, can be affected by stage of growth, age of the culture or oxygen concentration (Karsi and Lawrence, 2007). Other studies suggest that plasmids often become redundant in the absence of selective pressure and this causes them to become unstable, fragment or be completely eliminated (Jones et al., 1980). All biofilms prepared in this study were incubated in non-antibiotic-supplemented ASW/NSW. Therefore, an observed decrease in biofilm luminescence may also be due to the loss of pKluxCat.

Microbiological deposit details In accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, the Vibrio midae SY9 strain described in this specification was deposited with The Belgian Coordinated Collections of Microorganisms (BCCM) on 1 July 2013 under Accession No. LMG P-27727.

Acknowledgements Jacobsbaai Sea Products (Jacobsbaai, South Africa) and Aquafarm (Hermanus, South Africa) for providing abalone for this research. Dr Brett Macey and Dr Mark Cyrus at the DAFF Research Aquarium (Seapoint, South Africa) for assistance with diatom cultures. Karusha Moonsamy for constructing the bioluminescent plasmid, pKluxCat.

References Erasmus, J.H., Cook, PA. and Coyne, V.E., 1997. The role of bacteria in the digestion of seaweed by the abalone Haliotis midae. Aquaculture, 155(1), pp.377-386.

Hadi, J.A., Gutierrez, N., Alfaro, A.C. and Roberts, R.D., 2014. Use of probiotic bacteria to improve growth and survivability of farmed New Zealand abalone (Haliotis iris). N. Z. J. Mar. Freshw. Res. 48(3), pp.405-415.

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Stott, A.E., Takeuchi, T., Koike, Y. and Imada, 0., 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, 0., 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.

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Claims (13)

1. CLAIMS: A method of aquaculture of farmed abalone, the method comprising supplying at least Vibrio midae SY9 as a food source to post-larval abalone, wherein the Vibrio midae SY9 are supplied to the post-larval abalone on or within an alginate biofilm.
2. A method according to claim 1, wherein the biofilm is formed on a support.
3. 3. A method according to either of claims 1 or 2, wherein the concentration of the Vibrio midae SY9 in the biofilm is at least 108 cells/cm2.
4. 4. A method according to any one of claims 1 to 3, which further comprises supplying diatoms as an additional food source to the post-larval abalone.
5. 5. A method according to claim 4, wherein the concentration of the diatoms is at least 106 cells/cm2.
6. A method according to any one of claims 1 to 3, which does not comprise supplying diatoms as a food source to the post-larval abalone.
7. 7. A method according to any one of claims 1 to 6, which comprises replacing the biofilm after 5 or more days.
8. 8. A feed composition formulated for feeding post-larval abalone, the composition comprising Vibrio midae SY9 as a food source, wherein the Vibrio midae SY9 is coated onto or encapsulated within an alginate biofilm.
9. 9. A feed composition according to claim 8, wherein the biofilm is formed on a support.
10. 10. A feed composition according to either of claims 8 or 9, wherein the concentration of the Vibrio midae SY9 in the feed composition is at least 109 cells/ml.
11. 11. A feed composition according to any one of claims 8 to 10, which comprises diatoms as an additional food source for the post-larval abalone.
12. 12. A feed composition according to claim 11, wherein the concentration of the diatoms is at least 107 cells/ml.
13. 13. A feed composition according to any one of claims 8 to 12, which is stable in an aqueous composition for 5 or more days.