Oral administration of live probiotics such as Lactobacillus and Bifidobacterium spp. possess numerous beneficial effects. However, delivering viable probiotics to the host intestine has been a challenge due to poor survival of these bacteria during the gastric transit. An improved oral delivery system (modified alginate microcapsules) was developed in this study for targeted release of viable probiotics to the host intestine. Effect of various encapsulation parameters such as capsule size, alginate concentration, calcium chloride concentration, gelling/hardening time of microcapsules, addition of prebiotics and polymer coating, were individually investigated for improving the stability of microcapsules under simulated gastrointestinal (GI) conditions. Ability of microcapsules in protecting the viability of encapsulated bacteria improved significantly (p<0.05) in improving the stability of microcapsules. Optimisation of encapsulation parameters significantly improved the viability of encapsulated probiotics under simulated GI conditions. Furthermore, co-encapsulation of probiotics with complementary prebiotics (such as Hi-Maize starch) and chitosan coating provided additional protection to the encapsulated bacteria under simulated GI conditions. Release profile of chitosan-coated alginate-starch (CCAS) encapsulated bacteria was investigated in the GI tracts of different animal models. Addition of CCAS encapsulated bacteria to porcine GI contents (ex vivo) resulted in complete release of microencapsulated bacteria in the ileal contents within 8 h, while there was no significant release (p>0.05) of encapsulated bacteria in the gastric contents even after 24 h of incubation. In another experiment, CCAS microcapsules containing Lactobacillus casei Shirota (LCS) was orally administered to mice and the release profile of encapsulated bacteria was monitored throughout the murine GI tract for 24 h. Partial release of microencapsulated LCS was observed in duodenal and jejunal regions, while no significant (p>0.05) release of microencapsulated bacteria was observed in the stomach during the 24 h monitoring period. However, a significant release (nearly complete release) of microencapsulated bacteria was observed in ileal and colon of murine GI tract after 24 h. Elevated counts of LCS in ileum and colon indicated the most favorable site for the release of CCAS encapsulated bacteria. Further studies investigated the immunomodulatory activity of microencapsulated probiotic bacteria in a murine model. Lactobacillus casei Shirota was orally administered to mice either as microencapsulated or as free bacteria (non encapsulated) for two weeks. On day 14, the splenocytes from different experimental groups were harvested and assessed for ConA induced cytokine levels. A significant increase (p>0.05) in IFN-γ levels was observed in the activated splenocytes of groups treated with microencapsulated and free (non-encapsulated) LCS, compared to the control group (no LCS treatment). However, there was no significant difference (p<0.05) in the IFN-γ concentration between the groups treated with microencapsulated and free (non-encapsulated) LCS. No significant difference (p<0.05) in the IL-10 concentration was observed in the activated splenocytes of groups treated with microencapsulated and free (non-encapsulated) LCS. Finally, the stability of microencapsulated probiotics in different dairy products was investigated. CCAS microcapsules significantly protected the viability of probiotic bacteria in set and stirred yoghurts over 6-week refrigerated storage conditions compared to free (non-encapsulated) probiotics. Overall, chitosan-coated alginate-starch microcapsules developed in this study effectively protected the viability of probiotics from adverse gastric conditions and released the bacteria in the host intestine without detrimentally affecting its immunomodulatory properties. / Doctor of Philosophy

Probiotic bacteria such as Lactobacillus and Bifidobacterium species are normal inhabitants of healthy gastrointestinal (GI) tract, which may promote beneficial effects on host through limiting the growth of undesirable micro-organisms and modulating the immune system. In the present study, Lactobacillus and Bifidobacterium strains were screened for their in vitro acid and bile tolerance, autoaggregation, coaggregation and hydrophobic abilities to identify potential probiotic bacteria. Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei LAFTI L26 were selected based on their overall tolerance to in vitro acidic conditions to further investigate their influence on various immune functions and gene expression in mice. Immunofluorescent analysis of small intestine in mice fed with L. acidophilus or L. paracasei demonstrated an increase of immunoglobulin (Ig)-A, interleukin (IL)-10 and interferon (IFN)- producing cells compared to control mice. In summary, L. acidophilus and L. paracasei showed tolerance to various gastric conditions and bile salts. Lactobacillus acidophilus and L. paracasei enhanced gut and systemic immune functions, particularly non-specific and specific immune responses in normal and CT mice. Moreover, L. acidophilus regulated the genes involved in various biological functions in small bowel of normal and CT mice, which provided a basis in understanding the pathways through which these bacteria are beneficial to the host. / Doctor of Philosophy (PhD)

The Greenshell™ mussel (GSM), Perna canaliculus, industry in New Zealand (NZ) is the largest aquaculture sector in the country. In 2006, the export earnings were valued at US$145 million which represented 65% of NZ aquaculture earnings. Historically, and at present, GSM production involves the capture of wild mussels on ropes followed by on-growing of these animals to market size (approximately 14 months). However, hatchery production of GSM has been developed in recent years. Hatchery production will alleviate the seasonal uncertainties of current techniques and allow the benefits of selective breeding programs. To date, efforts to produce commercial quantities of GSM in hatcheries have been hampered by unreliable larval rearing. These problems were often alleviated by antibiotic use, which implied bacterial pathogens as the cause. Yet, the ongoing use of antibiotics is not sustainable because of increasing legislative restrictions on their use and the possible emergence of antibiotic resistant bacteria. Hence, the identification and use of novel probiotics was investigated as an alternative. Because of a lack of previous work, it was necessary to investigate the bacterial pathogenesis of GSM larvae in the initial stages and, hence, to determine the cause of disease against which the probiotics would be active. Twenty-two bacterial strains, isolated from compromised larvae, were screened for larval toxicity using a larval bioassay. Two strains were identified as potential pathogens. Sequencing of the 16S rRNA gene identified Vibrio splendidus and Vibrio sp. DO1, a Vibrio coralliilyticus/neptunius-like isolate, as pathogens of GSM larvae. These strains had the- ability to cause 83 and 75% GSM larval mortality in vitro respectively, at a concentration 102 CFU ml-1. Histopathology indicated the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. Although two bacterial pathogens were identified, the successful design and implementation of protective measures in the hatchery still required an understanding of the dynamics of the infection process. Developing an in situ experimental model for infection was therefore paramount. The minimum effective pathogenic dose (MEPD) of V. splendidus (105 CFU ml-1) and Vibrio sp. DO1 (106 CFU ml-1) was demonstrated for GSM larvae during hatchery production. In a flow-through water hatchery system, larvae given 1-2 hours of static water exposure with these pathogen doses, after which flowthrough processes resumed, averaged 58% and 69% cumulative mortality, respectively, on the fourth day following pathogen exposure. Larvae exposed to a dosage one order of magnitude greater than the MEPD, had higher mortalities of 73% and 96% for V. splendidus and Vibrio sp. DO1 respectively. These four levels of mortality were significantly greater than those of the non-exposed control larvae, averaging 23% in the experiments involving V. splendidus and 35% with Vibrio sp. DO1. Experiments were repeated four times to establish reproducibility. The infection models were reproducible and provided a tool to assess measures for the protection of GSM larvae against infection in the hatchery environment. A bioassay was developed to screen and select bacterial strains as potential probiotics for GSM larvae. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio sp. DO1 and V. splendidus were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls (p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 hours revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics. Following hatchery pilot trials, two probiotic strains were chosen for further study, namely strains 0444 and 0536. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately in a GSM hatchery facility during routine larval rearing and when the larvae were challenged with a high and low pathogenic dose of Vibrio sp. DO1 and V. splendidus. In all experiments, probiotic application significantly improved larval survival, if administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. If the probiotics were administered prior to pathogen challenge, larval survival averaged 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. In a repeat experiment, these benefits were reproduced, with the exception of A. macleodii 0444 trialled against V. splendidus. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This was the first time A. macleodii and Neptunomonas sp. were demonstrated as probiotic bacteria. Many studies document probiotic application in aquaculture under conditions of pathogen attack, yet few describe the use of probiotics during routine production. The effects of administering the probiotic, A. macleodii 0444, during routine GSM larvae production, were compared against larvae from the same cohort that were not treated with the probiotic. The probiotic was administered daily for the first 11 days of the larval period and was provided at two concentrations, 107 CFU ml-1 and 108 CFU ml-1. Measures of larval swimming activity, gut colouration, lipid levels, larval survival, larval size and settlement success were recorded. There were minimal differences in all parameters between larvae provided the probiotic and control larvae. Probiotic treated larvae consumed more food and had higher lipid levels at the end of the larval period, but these were not statistically significant. All treatments completed the larval phase and settled successfully after metamorphosis. Survival at the end of the larval period was 37.2%, 38.8%, and 34.8% for control, 107 CFU ml-1 and 108 CFU ml-1 treatments respectively. The probiotic was still detected in larvae seven days after the final addition to the tanks. Animals were further grown in the field at a commercial farm. The probiotic was not detected in mussels at four months after leaving the hatchery. Combination use of the two probiotics, A. macleodii 0444 and Neptunomonas sp. 0536, was investigated to determine whether additive protection against pathogen attack with Vibrio sp. DO1 and V. splendidus was afforded to GSM larvae. The effects of combination administration were compared with larvae administered each probiotic as single strains and non-inoculated larvae. Additionally, two concentrations were tested for each probiotic, both singly and in combination, 107 and 108 CFU ml-1. Larvae were administered probiotics daily for the first six days, challenged with pathogens on the third day and then reared until settlement (day 19). Although protection against pathogen attack was observed in combination treatments, when compared with single-strain administration, additive protection was not apparent. Administration of 108 CFU ml-1 levels of probiotics, both singly and in combination, afforded larval survival slightly better than 107 CFU ml-1 levels, although this was rarely statistically significant. On the other hand, the higher levels of probiotic led to smaller larvae and lower feed rates for the majority of the 19-day trial. At the end of the study, larval sizes were smaller in the treatment applied a combination of probiotics at 108 CFU ml-1 than those of the other treatments. Additionally, towards the end of the larval period, feed consumption in the combination 108 CFU ml-1 treatment was similar to that witnessed in the other probiotic treatments one day previously. This suggested that either the larvae were compromised or they were growing slower. Despite a lack of additive protection against a single strain pathogen attack being demonstrated, the potential benefit of multi-strain probiotics, as prophylactic measures against every-day microbial encounters in larviculture, would remain. Although 108 CFU ml-1 levels appeared to protect against pathogen attack slightly better, they were also potentially detrimental to normal larval rearing when administered in combination. Following the successful completion of the larval period and pathogen protection afforded with a combination of probiotics at 107 CFU ml-1, this level was recommended as the best concentration of each probiotic where combination administration would be applied. The work presented in this thesis supports the use of A. macleodii 0444 and Neptunomonas sp. 0536 in the routine rearing of GSM larvae. The ability to produce settled juvenile mussels, equal in numbers to those produced in normal healthy conditions, plus the benefits against pathogen attack led to the recommendation of their use on a routine prophylactic basis in GSM larval rearing. Their use for this purpose is intended in the near future. A provisional patent has been prepared and will be submitted shortly. It is anticipated that future work will continue with these probiotic strains to determine their potential benefit for other aquaculture species.

The Greenshell™ mussel (GSM), Perna canaliculus, industry in New Zealand (NZ) is the largest aquaculture sector in the country. In 2006, the export earnings were valued at US$145 million which represented 65% of NZ aquaculture earnings. Historically, and at present, GSM production involves the capture of wild mussels on ropes followed by on-growing of these animals to market size (approximately 14 months). However, hatchery production of GSM has been developed in recent years. Hatchery production will alleviate the seasonal uncertainties of current techniques and allow the benefits of selective breeding programs. To date, efforts to produce commercial quantities of GSM in hatcheries have been hampered by unreliable larval rearing. These problems were often alleviated by antibiotic use, which implied bacterial pathogens as the cause. Yet, the ongoing use of antibiotics is not sustainable because of increasing legislative restrictions on their use and the possible emergence of antibiotic resistant bacteria. Hence, the identification and use of novel probiotics was investigated as an alternative. Because of a lack of previous work, it was necessary to investigate the bacterial pathogenesis of GSM larvae in the initial stages and, hence, to determine the cause of disease against which the probiotics would be active. Twenty-two bacterial strains, isolated from compromised larvae, were screened for larval toxicity using a larval bioassay. Two strains were identified as potential pathogens. Sequencing of the 16S rRNA gene identified Vibrio splendidus and Vibrio sp. DO1, a Vibrio coralliilyticus/neptunius-like isolate, as pathogens of GSM larvae. These strains had the- ability to cause 83 and 75% GSM larval mortality in vitro respectively, at a concentration 102 CFU ml-1. Histopathology indicated the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. Although two bacterial pathogens were identified, the successful design and implementation of protective measures in the hatchery still required an understanding of the dynamics of the infection process. Developing an in situ experimental model for infection was therefore paramount. The minimum effective pathogenic dose (MEPD) of V. splendidus (105 CFU ml-1) and Vibrio sp. DO1 (106 CFU ml-1) was demonstrated for GSM larvae during hatchery production. In a flow-through water hatchery system, larvae given 1-2 hours of static water exposure with these pathogen doses, after which flowthrough processes resumed, averaged 58% and 69% cumulative mortality, respectively, on the fourth day following pathogen exposure. Larvae exposed to a dosage one order of magnitude greater than the MEPD, had higher mortalities of 73% and 96% for V. splendidus and Vibrio sp. DO1 respectively. These four levels of mortality were significantly greater than those of the non-exposed control larvae, averaging 23% in the experiments involving V. splendidus and 35% with Vibrio sp. DO1. Experiments were repeated four times to establish reproducibility. The infection models were reproducible and provided a tool to assess measures for the protection of GSM larvae against infection in the hatchery environment. A bioassay was developed to screen and select bacterial strains as potential probiotics for GSM larvae. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio sp. DO1 and V. splendidus were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls (p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 hours revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics. Following hatchery pilot trials, two probiotic strains were chosen for further study, namely strains 0444 and 0536. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately in a GSM hatchery facility during routine larval rearing and when the larvae were challenged with a high and low pathogenic dose of Vibrio sp. DO1 and V. splendidus. In all experiments, probiotic application significantly improved larval survival, if administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. If the probiotics were administered prior to pathogen challenge, larval survival averaged 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. In a repeat experiment, these benefits were reproduced, with the exception of A. macleodii 0444 trialled against V. splendidus. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This was the first time A. macleodii and Neptunomonas sp. were demonstrated as probiotic bacteria. Many studies document probiotic application in aquaculture under conditions of pathogen attack, yet few describe the use of probiotics during routine production. The effects of administering the probiotic, A. macleodii 0444, during routine GSM larvae production, were compared against larvae from the same cohort that were not treated with the probiotic. The probiotic was administered daily for the first 11 days of the larval period and was provided at two concentrations, 107 CFU ml-1 and 108 CFU ml-1. Measures of larval swimming activity, gut colouration, lipid levels, larval survival, larval size and settlement success were recorded. There were minimal differences in all parameters between larvae provided the probiotic and control larvae. Probiotic treated larvae consumed more food and had higher lipid levels at the end of the larval period, but these were not statistically significant. All treatments completed the larval phase and settled successfully after metamorphosis. Survival at the end of the larval period was 37.2%, 38.8%, and 34.8% for control, 107 CFU ml-1 and 108 CFU ml-1 treatments respectively. The probiotic was still detected in larvae seven days after the final addition to the tanks. Animals were further grown in the field at a commercial farm. The probiotic was not detected in mussels at four months after leaving the hatchery. Combination use of the two probiotics, A. macleodii 0444 and Neptunomonas sp. 0536, was investigated to determine whether additive protection against pathogen attack with Vibrio sp. DO1 and V. splendidus was afforded to GSM larvae. The effects of combination administration were compared with larvae administered each probiotic as single strains and non-inoculated larvae. Additionally, two concentrations were tested for each probiotic, both singly and in combination, 107 and 108 CFU ml-1. Larvae were administered probiotics daily for the first six days, challenged with pathogens on the third day and then reared until settlement (day 19). Although protection against pathogen attack was observed in combination treatments, when compared with single-strain administration, additive protection was not apparent. Administration of 108 CFU ml-1 levels of probiotics, both singly and in combination, afforded larval survival slightly better than 107 CFU ml-1 levels, although this was rarely statistically significant. On the other hand, the higher levels of probiotic led to smaller larvae and lower feed rates for the majority of the 19-day trial. At the end of the study, larval sizes were smaller in the treatment applied a combination of probiotics at 108 CFU ml-1 than those of the other treatments. Additionally, towards the end of the larval period, feed consumption in the combination 108 CFU ml-1 treatment was similar to that witnessed in the other probiotic treatments one day previously. This suggested that either the larvae were compromised or they were growing slower. Despite a lack of additive protection against a single strain pathogen attack being demonstrated, the potential benefit of multi-strain probiotics, as prophylactic measures against every-day microbial encounters in larviculture, would remain. Although 108 CFU ml-1 levels appeared to protect against pathogen attack slightly better, they were also potentially detrimental to normal larval rearing when administered in combination. Following the successful completion of the larval period and pathogen protection afforded with a combination of probiotics at 107 CFU ml-1, this level was recommended as the best concentration of each probiotic where combination administration would be applied. The work presented in this thesis supports the use of A. macleodii 0444 and Neptunomonas sp. 0536 in the routine rearing of GSM larvae. The ability to produce settled juvenile mussels, equal in numbers to those produced in normal healthy conditions, plus the benefits against pathogen attack led to the recommendation of their use on a routine prophylactic basis in GSM larval rearing. Their use for this purpose is intended in the near future. A provisional patent has been prepared and will be submitted shortly. It is anticipated that future work will continue with these probiotic strains to determine their potential benefit for other aquaculture species.

The Greenshell™ mussel (GSM), Perna canaliculus, industry in New Zealand (NZ) is the largest aquaculture sector in the country. In 2006, the export earnings were valued at US$145 million which represented 65% of NZ aquaculture earnings. Historically, and at present, GSM production involves the capture of wild mussels on ropes followed by on-growing of these animals to market size (approximately 14 months). However, hatchery production of GSM has been developed in recent years. Hatchery production will alleviate the seasonal uncertainties of current techniques and allow the benefits of selective breeding programs. To date, efforts to produce commercial quantities of GSM in hatcheries have been hampered by unreliable larval rearing. These problems were often alleviated by antibiotic use, which implied bacterial pathogens as the cause. Yet, the ongoing use of antibiotics is not sustainable because of increasing legislative restrictions on their use and the possible emergence of antibiotic resistant bacteria. Hence, the identification and use of novel probiotics was investigated as an alternative. Because of a lack of previous work, it was necessary to investigate the bacterial pathogenesis of GSM larvae in the initial stages and, hence, to determine the cause of disease against which the probiotics would be active. Twenty-two bacterial strains, isolated from compromised larvae, were screened for larval toxicity using a larval bioassay. Two strains were identified as potential pathogens. Sequencing of the 16S rRNA gene identified Vibrio splendidus and Vibrio sp. DO1, a Vibrio coralliilyticus/neptunius-like isolate, as pathogens of GSM larvae. These strains had the- ability to cause 83 and 75% GSM larval mortality in vitro respectively, at a concentration 102 CFU ml-1. Histopathology indicated the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. Although two bacterial pathogens were identified, the successful design and implementation of protective measures in the hatchery still required an understanding of the dynamics of the infection process. Developing an in situ experimental model for infection was therefore paramount. The minimum effective pathogenic dose (MEPD) of V. splendidus (105 CFU ml-1) and Vibrio sp. DO1 (106 CFU ml-1) was demonstrated for GSM larvae during hatchery production. In a flow-through water hatchery system, larvae given 1-2 hours of static water exposure with these pathogen doses, after which flowthrough processes resumed, averaged 58% and 69% cumulative mortality, respectively, on the fourth day following pathogen exposure. Larvae exposed to a dosage one order of magnitude greater than the MEPD, had higher mortalities of 73% and 96% for V. splendidus and Vibrio sp. DO1 respectively. These four levels of mortality were significantly greater than those of the non-exposed control larvae, averaging 23% in the experiments involving V. splendidus and 35% with Vibrio sp. DO1. Experiments were repeated four times to establish reproducibility. The infection models were reproducible and provided a tool to assess measures for the protection of GSM larvae against infection in the hatchery environment. A bioassay was developed to screen and select bacterial strains as potential probiotics for GSM larvae. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio sp. DO1 and V. splendidus were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls (p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 hours revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics. Following hatchery pilot trials, two probiotic strains were chosen for further study, namely strains 0444 and 0536. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately in a GSM hatchery facility during routine larval rearing and when the larvae were challenged with a high and low pathogenic dose of Vibrio sp. DO1 and V. splendidus. In all experiments, probiotic application significantly improved larval survival, if administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. If the probiotics were administered prior to pathogen challenge, larval survival averaged 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. In a repeat experiment, these benefits were reproduced, with the exception of A. macleodii 0444 trialled against V. splendidus. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This was the first time A. macleodii and Neptunomonas sp. were demonstrated as probiotic bacteria. Many studies document probiotic application in aquaculture under conditions of pathogen attack, yet few describe the use of probiotics during routine production. The effects of administering the probiotic, A. macleodii 0444, during routine GSM larvae production, were compared against larvae from the same cohort that were not treated with the probiotic. The probiotic was administered daily for the first 11 days of the larval period and was provided at two concentrations, 107 CFU ml-1 and 108 CFU ml-1. Measures of larval swimming activity, gut colouration, lipid levels, larval survival, larval size and settlement success were recorded. There were minimal differences in all parameters between larvae provided the probiotic and control larvae. Probiotic treated larvae consumed more food and had higher lipid levels at the end of the larval period, but these were not statistically significant. All treatments completed the larval phase and settled successfully after metamorphosis. Survival at the end of the larval period was 37.2%, 38.8%, and 34.8% for control, 107 CFU ml-1 and 108 CFU ml-1 treatments respectively. The probiotic was still detected in larvae seven days after the final addition to the tanks. Animals were further grown in the field at a commercial farm. The probiotic was not detected in mussels at four months after leaving the hatchery. Combination use of the two probiotics, A. macleodii 0444 and Neptunomonas sp. 0536, was investigated to determine whether additive protection against pathogen attack with Vibrio sp. DO1 and V. splendidus was afforded to GSM larvae. The effects of combination administration were compared with larvae administered each probiotic as single strains and non-inoculated larvae. Additionally, two concentrations were tested for each probiotic, both singly and in combination, 107 and 108 CFU ml-1. Larvae were administered probiotics daily for the first six days, challenged with pathogens on the third day and then reared until settlement (day 19). Although protection against pathogen attack was observed in combination treatments, when compared with single-strain administration, additive protection was not apparent. Administration of 108 CFU ml-1 levels of probiotics, both singly and in combination, afforded larval survival slightly better than 107 CFU ml-1 levels, although this was rarely statistically significant. On the other hand, the higher levels of probiotic led to smaller larvae and lower feed rates for the majority of the 19-day trial. At the end of the study, larval sizes were smaller in the treatment applied a combination of probiotics at 108 CFU ml-1 than those of the other treatments. Additionally, towards the end of the larval period, feed consumption in the combination 108 CFU ml-1 treatment was similar to that witnessed in the other probiotic treatments one day previously. This suggested that either the larvae were compromised or they were growing slower. Despite a lack of additive protection against a single strain pathogen attack being demonstrated, the potential benefit of multi-strain probiotics, as prophylactic measures against every-day microbial encounters in larviculture, would remain. Although 108 CFU ml-1 levels appeared to protect against pathogen attack slightly better, they were also potentially detrimental to normal larval rearing when administered in combination. Following the successful completion of the larval period and pathogen protection afforded with a combination of probiotics at 107 CFU ml-1, this level was recommended as the best concentration of each probiotic where combination administration would be applied. The work presented in this thesis supports the use of A. macleodii 0444 and Neptunomonas sp. 0536 in the routine rearing of GSM larvae. The ability to produce settled juvenile mussels, equal in numbers to those produced in normal healthy conditions, plus the benefits against pathogen attack led to the recommendation of their use on a routine prophylactic basis in GSM larval rearing. Their use for this purpose is intended in the near future. A provisional patent has been prepared and will be submitted shortly. It is anticipated that future work will continue with these probiotic strains to determine their potential benefit for other aquaculture species.

Starter cultures of different bacteria strains like lactic acid producing bacteria, Staphylococcus and Kocuria are used when making salami. Starter cultures give the sausage specific flavours and improve the quality and ripening of the final product. Probiotic strains can also be added during the production of salami. Studies have shown that probiotics are good for health and are therefore added to food, such as fermented sausages. In order to work as a probiotic strain, the bacteria have to survive during the production process, storage and through the whole human gastrointestinal tract. The aim of this study was to trace the probiotic strains Lactobacillus casei and Lactobacillus paracasei in salami samples to see if they had survived the production process. Methods used were DNA extraction, PCR, colony PCR and gel electrophoresis. Out of 100 samples in duplicate run in PCR, probiotics were found in only 3 of them. To see if screening of probiotics directly from plates was possible, a colony PCR was done. Colony PCR was made on colonies of two different strains of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus sakei. From each bacteria strain, 5 colonies were analysed. Result showed that colony PCR, to screen for probiotic is a possible method.

Humans harbour a diverse suite of microorganisms in and on their bodies. These microorganisms collectively amount to 10 times more cells than human cells in the body, and their combined genomes have more than 100 times more genes than the human genome does. Despite our understanding of the composition, diversity, and abundance of microorganisms of the human body, it is surprising how little we know about the structure and function of the human microbiome. Here, I use network structure to describe interactions among human-associated microbiota and the human body by exploring differences in structure of human microbiomes across five regions of the body and the robustness of these networks to perturbations. My results show that positive interactions among microbiota are extremely important in structuring microbiome networks and those structural aspects of microbiome networks play a major role in their response to perturbations.

The Greenshell™ mussel (GSM), Perna canaliculus, industry in New Zealand (NZ) is the largest aquaculture sector in the country. In 2006, the export earnings were valued at US$145 million which represented 65% of NZ aquaculture earnings. Historically, and at present, GSM production involves the capture of wild mussels on ropes followed by on-growing of these animals to market size (approximately 14 months). However, hatchery production of GSM has been developed in recent years. Hatchery production will alleviate the seasonal uncertainties of current techniques and allow the benefits of selective breeding programs. To date, efforts to produce commercial quantities of GSM in hatcheries have been hampered by unreliable larval rearing. These problems were often alleviated by antibiotic use, which implied bacterial pathogens as the cause. Yet, the ongoing use of antibiotics is not sustainable because of increasing legislative restrictions on their use and the possible emergence of antibiotic resistant bacteria. Hence, the identification and use of novel probiotics was investigated as an alternative. Because of a lack of previous work, it was necessary to investigate the bacterial pathogenesis of GSM larvae in the initial stages and, hence, to determine the cause of disease against which the probiotics would be active. Twenty-two bacterial strains, isolated from compromised larvae, were screened for larval toxicity using a larval bioassay. Two strains were identified as potential pathogens. Sequencing of the 16S rRNA gene identified Vibrio splendidus and Vibrio sp. DO1, a Vibrio coralliilyticus/neptunius-like isolate, as pathogens of GSM larvae. These strains had the- ability to cause 83 and 75% GSM larval mortality in vitro respectively, at a concentration 102 CFU ml-1. Histopathology indicated the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. Although two bacterial pathogens were identified, the successful design and implementation of protective measures in the hatchery still required an understanding of the dynamics of the infection process. Developing an in situ experimental model for infection was therefore paramount. The minimum effective pathogenic dose (MEPD) of V. splendidus (105 CFU ml-1) and Vibrio sp. DO1 (106 CFU ml-1) was demonstrated for GSM larvae during hatchery production. In a flow-through water hatchery system, larvae given 1-2 hours of static water exposure with these pathogen doses, after which flowthrough processes resumed, averaged 58% and 69% cumulative mortality, respectively, on the fourth day following pathogen exposure. Larvae exposed to a dosage one order of magnitude greater than the MEPD, had higher mortalities of 73% and 96% for V. splendidus and Vibrio sp. DO1 respectively. These four levels of mortality were significantly greater than those of the non-exposed control larvae, averaging 23% in the experiments involving V. splendidus and 35% with Vibrio sp. DO1. Experiments were repeated four times to establish reproducibility. The infection models were reproducible and provided a tool to assess measures for the protection of GSM larvae against infection in the hatchery environment. A bioassay was developed to screen and select bacterial strains as potential probiotics for GSM larvae. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio sp. DO1 and V. splendidus were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls (p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 hours revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics. Following hatchery pilot trials, two probiotic strains were chosen for further study, namely strains 0444 and 0536. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately in a GSM hatchery facility during routine larval rearing and when the larvae were challenged with a high and low pathogenic dose of Vibrio sp. DO1 and V. splendidus. In all experiments, probiotic application significantly improved larval survival, if administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. If the probiotics were administered prior to pathogen challenge, larval survival averaged 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. In a repeat experiment, these benefits were reproduced, with the exception of A. macleodii 0444 trialled against V. splendidus. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This was the first time A. macleodii and Neptunomonas sp. were demonstrated as probiotic bacteria. Many studies document probiotic application in aquaculture under conditions of pathogen attack, yet few describe the use of probiotics during routine production. The effects of administering the probiotic, A. macleodii 0444, during routine GSM larvae production, were compared against larvae from the same cohort that were not treated with the probiotic. The probiotic was administered daily for the first 11 days of the larval period and was provided at two concentrations, 107 CFU ml-1 and 108 CFU ml-1. Measures of larval swimming activity, gut colouration, lipid levels, larval survival, larval size and settlement success were recorded. There were minimal differences in all parameters between larvae provided the probiotic and control larvae. Probiotic treated larvae consumed more food and had higher lipid levels at the end of the larval period, but these were not statistically significant. All treatments completed the larval phase and settled successfully after metamorphosis. Survival at the end of the larval period was 37.2%, 38.8%, and 34.8% for control, 107 CFU ml-1 and 108 CFU ml-1 treatments respectively. The probiotic was still detected in larvae seven days after the final addition to the tanks. Animals were further grown in the field at a commercial farm. The probiotic was not detected in mussels at four months after leaving the hatchery. Combination use of the two probiotics, A. macleodii 0444 and Neptunomonas sp. 0536, was investigated to determine whether additive protection against pathogen attack with Vibrio sp. DO1 and V. splendidus was afforded to GSM larvae. The effects of combination administration were compared with larvae administered each probiotic as single strains and non-inoculated larvae. Additionally, two concentrations were tested for each probiotic, both singly and in combination, 107 and 108 CFU ml-1. Larvae were administered probiotics daily for the first six days, challenged with pathogens on the third day and then reared until settlement (day 19). Although protection against pathogen attack was observed in combination treatments, when compared with single-strain administration, additive protection was not apparent. Administration of 108 CFU ml-1 levels of probiotics, both singly and in combination, afforded larval survival slightly better than 107 CFU ml-1 levels, although this was rarely statistically significant. On the other hand, the higher levels of probiotic led to smaller larvae and lower feed rates for the majority of the 19-day trial. At the end of the study, larval sizes were smaller in the treatment applied a combination of probiotics at 108 CFU ml-1 than those of the other treatments. Additionally, towards the end of the larval period, feed consumption in the combination 108 CFU ml-1 treatment was similar to that witnessed in the other probiotic treatments one day previously. This suggested that either the larvae were compromised or they were growing slower. Despite a lack of additive protection against a single strain pathogen attack being demonstrated, the potential benefit of multi-strain probiotics, as prophylactic measures against every-day microbial encounters in larviculture, would remain. Although 108 CFU ml-1 levels appeared to protect against pathogen attack slightly better, they were also potentially detrimental to normal larval rearing when administered in combination. Following the successful completion of the larval period and pathogen protection afforded with a combination of probiotics at 107 CFU ml-1, this level was recommended as the best concentration of each probiotic where combination administration would be applied. The work presented in this thesis supports the use of A. macleodii 0444 and Neptunomonas sp. 0536 in the routine rearing of GSM larvae. The ability to produce settled juvenile mussels, equal in numbers to those produced in normal healthy conditions, plus the benefits against pathogen attack led to the recommendation of their use on a routine prophylactic basis in GSM larval rearing. Their use for this purpose is intended in the near future. A provisional patent has been prepared and will be submitted shortly. It is anticipated that future work will continue with these probiotic strains to determine their potential benefit for other aquaculture species.

The Greenshell™ mussel (GSM), Perna canaliculus, industry in New Zealand (NZ) is the largest aquaculture sector in the country. In 2006, the export earnings were valued at US$145 million which represented 65% of NZ aquaculture earnings. Historically, and at present, GSM production involves the capture of wild mussels on ropes followed by on-growing of these animals to market size (approximately 14 months). However, hatchery production of GSM has been developed in recent years. Hatchery production will alleviate the seasonal uncertainties of current techniques and allow the benefits of selective breeding programs. To date, efforts to produce commercial quantities of GSM in hatcheries have been hampered by unreliable larval rearing. These problems were often alleviated by antibiotic use, which implied bacterial pathogens as the cause. Yet, the ongoing use of antibiotics is not sustainable because of increasing legislative restrictions on their use and the possible emergence of antibiotic resistant bacteria. Hence, the identification and use of novel probiotics was investigated as an alternative. Because of a lack of previous work, it was necessary to investigate the bacterial pathogenesis of GSM larvae in the initial stages and, hence, to determine the cause of disease against which the probiotics would be active. Twenty-two bacterial strains, isolated from compromised larvae, were screened for larval toxicity using a larval bioassay. Two strains were identified as potential pathogens. Sequencing of the 16S rRNA gene identified Vibrio splendidus and Vibrio sp. DO1, a Vibrio coralliilyticus/neptunius-like isolate, as pathogens of GSM larvae. These strains had the- ability to cause 83 and 75% GSM larval mortality in vitro respectively, at a concentration 102 CFU ml-1. Histopathology indicated the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. Although two bacterial pathogens were identified, the successful design and implementation of protective measures in the hatchery still required an understanding of the dynamics of the infection process. Developing an in situ experimental model for infection was therefore paramount. The minimum effective pathogenic dose (MEPD) of V. splendidus (105 CFU ml-1) and Vibrio sp. DO1 (106 CFU ml-1) was demonstrated for GSM larvae during hatchery production. In a flow-through water hatchery system, larvae given 1-2 hours of static water exposure with these pathogen doses, after which flowthrough processes resumed, averaged 58% and 69% cumulative mortality, respectively, on the fourth day following pathogen exposure. Larvae exposed to a dosage one order of magnitude greater than the MEPD, had higher mortalities of 73% and 96% for V. splendidus and Vibrio sp. DO1 respectively. These four levels of mortality were significantly greater than those of the non-exposed control larvae, averaging 23% in the experiments involving V. splendidus and 35% with Vibrio sp. DO1. Experiments were repeated four times to establish reproducibility. The infection models were reproducible and provided a tool to assess measures for the protection of GSM larvae against infection in the hatchery environment. A bioassay was developed to screen and select bacterial strains as potential probiotics for GSM larvae. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio sp. DO1 and V. splendidus were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls (p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 hours revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics. Following hatchery pilot trials, two probiotic strains were chosen for further study, namely strains 0444 and 0536. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately in a GSM hatchery facility during routine larval rearing and when the larvae were challenged with a high and low pathogenic dose of Vibrio sp. DO1 and V. splendidus. In all experiments, probiotic application significantly improved larval survival, if administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. If the probiotics were administered prior to pathogen challenge, larval survival averaged 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. In a repeat experiment, these benefits were reproduced, with the exception of A. macleodii 0444 trialled against V. splendidus. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This was the first time A. macleodii and Neptunomonas sp. were demonstrated as probiotic bacteria. Many studies document probiotic application in aquaculture under conditions of pathogen attack, yet few describe the use of probiotics during routine production. The effects of administering the probiotic, A. macleodii 0444, during routine GSM larvae production, were compared against larvae from the same cohort that were not treated with the probiotic. The probiotic was administered daily for the first 11 days of the larval period and was provided at two concentrations, 107 CFU ml-1 and 108 CFU ml-1. Measures of larval swimming activity, gut colouration, lipid levels, larval survival, larval size and settlement success were recorded. There were minimal differences in all parameters between larvae provided the probiotic and control larvae. Probiotic treated larvae consumed more food and had higher lipid levels at the end of the larval period, but these were not statistically significant. All treatments completed the larval phase and settled successfully after metamorphosis. Survival at the end of the larval period was 37.2%, 38.8%, and 34.8% for control, 107 CFU ml-1 and 108 CFU ml-1 treatments respectively. The probiotic was still detected in larvae seven days after the final addition to the tanks. Animals were further grown in the field at a commercial farm. The probiotic was not detected in mussels at four months after leaving the hatchery. Combination use of the two probiotics, A. macleodii 0444 and Neptunomonas sp. 0536, was investigated to determine whether additive protection against pathogen attack with Vibrio sp. DO1 and V. splendidus was afforded to GSM larvae. The effects of combination administration were compared with larvae administered each probiotic as single strains and non-inoculated larvae. Additionally, two concentrations were tested for each probiotic, both singly and in combination, 107 and 108 CFU ml-1. Larvae were administered probiotics daily for the first six days, challenged with pathogens on the third day and then reared until settlement (day 19). Although protection against pathogen attack was observed in combination treatments, when compared with single-strain administration, additive protection was not apparent. Administration of 108 CFU ml-1 levels of probiotics, both singly and in combination, afforded larval survival slightly better than 107 CFU ml-1 levels, although this was rarely statistically significant. On the other hand, the higher levels of probiotic led to smaller larvae and lower feed rates for the majority of the 19-day trial. At the end of the study, larval sizes were smaller in the treatment applied a combination of probiotics at 108 CFU ml-1 than those of the other treatments. Additionally, towards the end of the larval period, feed consumption in the combination 108 CFU ml-1 treatment was similar to that witnessed in the other probiotic treatments one day previously. This suggested that either the larvae were compromised or they were growing slower. Despite a lack of additive protection against a single strain pathogen attack being demonstrated, the potential benefit of multi-strain probiotics, as prophylactic measures against every-day microbial encounters in larviculture, would remain. Although 108 CFU ml-1 levels appeared to protect against pathogen attack slightly better, they were also potentially detrimental to normal larval rearing when administered in combination. Following the successful completion of the larval period and pathogen protection afforded with a combination of probiotics at 107 CFU ml-1, this level was recommended as the best concentration of each probiotic where combination administration would be applied. The work presented in this thesis supports the use of A. macleodii 0444 and Neptunomonas sp. 0536 in the routine rearing of GSM larvae. The ability to produce settled juvenile mussels, equal in numbers to those produced in normal healthy conditions, plus the benefits against pathogen attack led to the recommendation of their use on a routine prophylactic basis in GSM larval rearing. Their use for this purpose is intended in the near future. A provisional patent has been prepared and will be submitted shortly. It is anticipated that future work will continue with these probiotic strains to determine their potential benefit for other aquaculture species.

The Greenshell™ mussel (GSM), Perna canaliculus, industry in New Zealand (NZ) is the largest aquaculture sector in the country. In 2006, the export earnings were valued at US$145 million which represented 65% of NZ aquaculture earnings. Historically, and at present, GSM production involves the capture of wild mussels on ropes followed by on-growing of these animals to market size (approximately 14 months). However, hatchery production of GSM has been developed in recent years. Hatchery production will alleviate the seasonal uncertainties of current techniques and allow the benefits of selective breeding programs. To date, efforts to produce commercial quantities of GSM in hatcheries have been hampered by unreliable larval rearing. These problems were often alleviated by antibiotic use, which implied bacterial pathogens as the cause. Yet, the ongoing use of antibiotics is not sustainable because of increasing legislative restrictions on their use and the possible emergence of antibiotic resistant bacteria. Hence, the identification and use of novel probiotics was investigated as an alternative. Because of a lack of previous work, it was necessary to investigate the bacterial pathogenesis of GSM larvae in the initial stages and, hence, to determine the cause of disease against which the probiotics would be active. Twenty-two bacterial strains, isolated from compromised larvae, were screened for larval toxicity using a larval bioassay. Two strains were identified as potential pathogens. Sequencing of the 16S rRNA gene identified Vibrio splendidus and Vibrio sp. DO1, a Vibrio coralliilyticus/neptunius-like isolate, as pathogens of GSM larvae. These strains had the- ability to cause 83 and 75% GSM larval mortality in vitro respectively, at a concentration 102 CFU ml-1. Histopathology indicated the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. Although two bacterial pathogens were identified, the successful design and implementation of protective measures in the hatchery still required an understanding of the dynamics of the infection process. Developing an in situ experimental model for infection was therefore paramount. The minimum effective pathogenic dose (MEPD) of V. splendidus (105 CFU ml-1) and Vibrio sp. DO1 (106 CFU ml-1) was demonstrated for GSM larvae during hatchery production. In a flow-through water hatchery system, larvae given 1-2 hours of static water exposure with these pathogen doses, after which flowthrough processes resumed, averaged 58% and 69% cumulative mortality, respectively, on the fourth day following pathogen exposure. Larvae exposed to a dosage one order of magnitude greater than the MEPD, had higher mortalities of 73% and 96% for V. splendidus and Vibrio sp. DO1 respectively. These four levels of mortality were significantly greater than those of the non-exposed control larvae, averaging 23% in the experiments involving V. splendidus and 35% with Vibrio sp. DO1. Experiments were repeated four times to establish reproducibility. The infection models were reproducible and provided a tool to assess measures for the protection of GSM larvae against infection in the hatchery environment. A bioassay was developed to screen and select bacterial strains as potential probiotics for GSM larvae. Sixty-nine isolates originating from a GSM hatchery environment were tested for probiotic activity in larval pathogen-challenge bioassays conducted in tissue culture dishes (TCDs). Vibrio sp. DO1 and V. splendidus were the tested pathogens. Forty of the tested isolates afforded larval survival significantly greater than pathogen controls (p < 0.05). The bioassay technique achieved a 58% success rate in searching for putative probiotics and highlighted the benefit of including the host animal in the first stage of the screening procedure. The time of inoculation of putative probiotic strains prior to pathogen challenge influenced the outcome of the assay. A pre-exposure period of 20 hours revealed a greater number of potential probiotics than a two-hour pre-exposure period. Pilot challenge tests, under normal hatchery conditions, confirmed the usefulness of the TCD screening method in recognising effective probiotics. Following hatchery pilot trials, two probiotic strains were chosen for further study, namely strains 0444 and 0536. Sequencing of the 16S rRNA gene and phylogenetic analysis identified the strains as Alteromonas macleodii 0444 and Neptunomonas sp. 0536. Both probiotics were evaluated separately in a GSM hatchery facility during routine larval rearing and when the larvae were challenged with a high and low pathogenic dose of Vibrio sp. DO1 and V. splendidus. In all experiments, probiotic application significantly improved larval survival, if administered prior to pathogen exposure. Across all experiments, larvae that were exposed to the high and low dosages of pathogens averaged 14% and 36% survival respectively on the fourth day following pathogen exposure. If the probiotics were administered prior to pathogen challenge, larval survival averaged 50% and 66% respectively. Non-inoculated control larvae and larvae administered the probiotic alone demonstrated 67% and 79% survival respectively. In a repeat experiment, these benefits were reproduced, with the exception of A. macleodii 0444 trialled against V. splendidus. Neptunomonas sp. 0536 appeared to suppress naturally occurring vibrios in the culture environment of healthy GSM larvae. This was the first time A. macleodii and Neptunomonas sp. were demonstrated as probiotic bacteria. Many studies document probiotic application in aquaculture under conditions of pathogen attack, yet few describe the use of probiotics during routine production. The effects of administering the probiotic, A. macleodii 0444, during routine GSM larvae production, were compared against larvae from the same cohort that were not treated with the probiotic. The probiotic was administered daily for the first 11 days of the larval period and was provided at two concentrations, 107 CFU ml-1 and 108 CFU ml-1. Measures of larval swimming activity, gut colouration, lipid levels, larval survival, larval size and settlement success were recorded. There were minimal differences in all parameters between larvae provided the probiotic and control larvae. Probiotic treated larvae consumed more food and had higher lipid levels at the end of the larval period, but these were not statistically significant. All treatments completed the larval phase and settled successfully after metamorphosis. Survival at the end of the larval period was 37.2%, 38.8%, and 34.8% for control, 107 CFU ml-1 and 108 CFU ml-1 treatments respectively. The probiotic was still detected in larvae seven days after the final addition to the tanks. Animals were further grown in the field at a commercial farm. The probiotic was not detected in mussels at four months after leaving the hatchery. Combination use of the two probiotics, A. macleodii 0444 and Neptunomonas sp. 0536, was investigated to determine whether additive protection against pathogen attack with Vibrio sp. DO1 and V. splendidus was afforded to GSM larvae. The effects of combination administration were compared with larvae administered each probiotic as single strains and non-inoculated larvae. Additionally, two concentrations were tested for each probiotic, both singly and in combination, 107 and 108 CFU ml-1. Larvae were administered probiotics daily for the first six days, challenged with pathogens on the third day and then reared until settlement (day 19). Although protection against pathogen attack was observed in combination treatments, when compared with single-strain administration, additive protection was not apparent. Administration of 108 CFU ml-1 levels of probiotics, both singly and in combination, afforded larval survival slightly better than 107 CFU ml-1 levels, although this was rarely statistically significant. On the other hand, the higher levels of probiotic led to smaller larvae and lower feed rates for the majority of the 19-day trial. At the end of the study, larval sizes were smaller in the treatment applied a combination of probiotics at 108 CFU ml-1 than those of the other treatments. Additionally, towards the end of the larval period, feed consumption in the combination 108 CFU ml-1 treatment was similar to that witnessed in the other probiotic treatments one day previously. This suggested that either the larvae were compromised or they were growing slower. Despite a lack of additive protection against a single strain pathogen attack being demonstrated, the potential benefit of multi-strain probiotics, as prophylactic measures against every-day microbial encounters in larviculture, would remain. Although 108 CFU ml-1 levels appeared to protect against pathogen attack slightly better, they were also potentially detrimental to normal larval rearing when administered in combination. Following the successful completion of the larval period and pathogen protection afforded with a combination of probiotics at 107 CFU ml-1, this level was recommended as the best concentration of each probiotic where combination administration would be applied. The work presented in this thesis supports the use of A. macleodii 0444 and Neptunomonas sp. 0536 in the routine rearing of GSM larvae. The ability to produce settled juvenile mussels, equal in numbers to those produced in normal healthy conditions, plus the benefits against pathogen attack led to the recommendation of their use on a routine prophylactic basis in GSM larval rearing. Their use for this purpose is intended in the near future. A provisional patent has been prepared and will be submitted shortly. It is anticipated that future work will continue with these probiotic strains to determine their potential benefit for other aquaculture species.