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Orientador: Antonio Fernando Monteiro Camargo / Coorientador: Daercy Maria Monteiro de Rezende Ayroza / Banca: Matheus Nicolino Peixoto Henares / Banca: Gianmarco Silva David / Resumo: Objetivou-se avaliar a influência das variáveis limnológicas e da velocidade da corrente no desenvolvimento de Limnoperna fortunei e no recrutamento de novos indivíduos em áreas com tanques-rede, no Reservatório Canoas II, no Rio Paranapanema. Mensalmente de agosto/2016 a janeiro/2017 em campo foram realizadas medições de: transparência, oxigênio dissolvido potencial hidrogeniônico, condutividade elétrica e sólidos totais dissolvidos. Além destes, a água foi coletada e armazenada em frascos para serem analisadas em laboratório: nitrogênio total, nitrito, nitrato, amônia, fósforo total, ortofosfato, cálcio, alcalinidade, dureza e clorofila a. Medições de velocidade da corrente da água foram obtidas utilizando um fluxômetro e um ADCP. As amostras de larvas de L. fortunei foram obtidas utilizando bomba de sucção e rede de plâncton de 40 μm. Foram confeccionados substratos de madeira em cruz para contagem e medição do tamanho de larvas fixados. As variáveis limnológicas foram investigadas por meio da Análise de Componentes Principais (ACP). A comparação entre o comprimento dos indivíduos nas duas áreas avaliadas e a medição da velocidade de corrente com o ADCP foi realizada por meio de Box Whisker Plot. Os resultados de velocidade de corrente da água com o fluxômetro e a da relação entre densidade de larvas na coluna d'água e número de indivíduos fixados foi feita com o auxílio de gráficos. As variáveis limnológicas podem ter influenciado no desenvolvimento do L. fortunei, princi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the influence of limnological variables and current velocity on the development of Limnoperna fortunei and the recruitment of new individuals in areas with network tanks in the Canoas II Reservoir on the Paranapanema River. Monthly from August / 2016 to January / 2017 in the field measurements were made of: transparency, dissolved oxygen potential hydrogen, electrical conductivity and total dissolved solids. In addition, water was collected and stored in bottles to be analyzed in laboratory: total nitrogen, nitrite, nitrate, ammonia, total phosphorus, orthophosphate, calcium, alkalinity, hardness and chlorophyll a. Water current velocity measurements were obtained using a flow meter and an ADCP. Samples of larvae of L. fortunei were obtained using a suction pump and 40 μm plankton net. Crosswood substrates were made for counting and measuring the size of fixed larvae. Limnological variables were investigated through Principal Component Analysis (PCA). The comparison between the length of the individuals in the two areas evaluated and the current velocity measurement with the ADCP was done by means of Box Whisker Plot. The results of current velocity of the water with the flowmeter and of the relation between larval density in the water column and number of fixed individuals was done with the aid of graphs. Limnological variables may have influenced the development of L. fortunei, especially in TR2. It has not been proven that water ... (Complete abstract click electronic access below) / Mestre

Invasive colonial tunicates pose substantial economic threat to the shellfish aquaculture industry, but their population dynamics and ecological impacts are highly variable and region-specific. This thesis contributes to our regional understanding of two such tunicates in Oregon. The first chapter explores the population dynamics of Didemnum vexillum, one of Oregon’s top 100 most dangerous invasive species, at an oyster farm. From May 2011 to 2016 the population fluctuated extensively, though did not exhibit any net growth over the study period. In the second chapter, I demonstrate that Botrylloides violaceus had no impact on the growth, condition, or organic composition of oysters and mussels grown in a simulation of longline aquaculture. Together, these studies paint a cautiously positive outlook for the shellfish aquaculture industry in Oregon. This thesis includes previously unpublished co-authored material.

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.

Invasive organisms are one of the major threats to the ecological integrity of aquatic systems in the 21st century. Among the most notorious and important aquatic invasive organisms are the dreissenid mussels, Dreissena polymorpha and D. rostriformis bugensis, which having originated in the Ponto-Caspian region are now common in many parts of Europe and North America. Dreissenids have large impacts on many aspects of lentic ecosystem functioning, the sum of which is thought to lead to the translocation of biological production from the pelagic to the littoral zones of lakes. In this thesis I explore the effects of dreissenids on the nearshore zones of large lakes, investigate the mechanisms by which dreissenids couple the pelagic and nearshore zones of lakes and attempt to elucidate the factors affecting the strength of the dreissenid-mediated connection between the pelagic and littoral zones. The effects of invasive organisms on an aquatic ecosystem will depend, in part, on the distribution and biomass of the invasive organisms in the system. In chapter 2 I present the results of a lake-wide survey of the distribution of invasive dreissenid mussels in Lake Simcoe, Ontario and discuss some of the factors that shape their distribution pattern in the lake. Dreissenid biomass averaged 27.2 (±24.3 SD) g shell-free dry mass (SFDM)/m2 in the main basin of Lake Simcoe and 12.4 (±16.9 SD) g SFDM/m2 in macrophyte-dominated Cook’s Bay. I argue that water movement is an important determinant of dreissenid distribution, both through catastrophic disturbance in shallow water and through non-catastrophic effects on substrate distribution and possibly food supply rates. In areas of dense macrophyte growth, mussel abundance was shown to be associated with that of preferred macrophyte taxa, in particular with that of Ceratophyllum demersum. I used the results of my survey and the relationships between environmental variables and dreissenid biomass to estimate the total biomass of dreissenids in Lake Simcoe: 12,000 tonnes SFDM. Most of the dreissenid biomass in Lake Simcoe was concentrated in the nearshore zone, where dreissenids would have maximal impacts on littoral biological production. One of the effects of the dreissenid invasion into the Laurentian Great Lakes appears to be a resurgence in the abundance of the nuisance alga Cladophora glomerata which experienced a marked decline following phosphorus abatement in the late 1970s and early 1980s. A subsidy of bioavailable phosphorus excreted by dreissenid mussels could be an important mechanism facilitating the growth of C. glomerata. In chapter 3, I describe a survey of dreissenid distribution and abundance followed by in situ experiments designed to measure dreissenid phosphorus excretion rates. Average dreissenid mussel abundance in our study area was 3674 (±2233 SD) individuals/m2, with an average biomass of 52.2 (±29.0 SD) g of shell free dry mass/m2. The mussels excreted bioavailable soluble reactive phosphorus (SRP) at an average rate of 7.0 μg SRP/g shell free dry mass/hour, contributing about 11 tonnes of SRP to the study area over the C. glomerata growing season. Dreissenids appear to be an important source of recycled bioavailable phosphorus to the littoral zone, potentially supplying more soluble reactive phosphorus to the study area than local watercourses and waste water treatment plants, and more phosphorus than is required to sustain local C. glomerata growth. Dreissenid establishment in many systems coincides with increases in the abundance and diversity of littoral benthic invertebrates and with changes to community composition of the benthos. Currently, there is a lack of long-term studies of the impact of dreissenid mussels on hard-substrate inhabiting littoral benthos. In chapter 4 I compare the littoral benthos of Lake Simcoe, Ontario just prior, and 14 years following the establishment of dreissenids in the lake. Densities of non-dreissenid invertebrates on hard substrata increased by nearly 50 times, from an average of 367.9 (±460.8 SD) individuals/m2 in 1993 to an average of 16,706.4 (±10,204.5 SD) individuals/m2 in 2008. The taxonomic diversity of the benthos increased significantly. The distribution of benthic organisms also changed; the numerical abundance of benthos has become more even across depths and sites, as has community composition. I suggest that in addition to increasing resource availability to benthic organisms dreissenids have also caused a homogenization of the littoral habitat by increasing the evenness of the distribution of food and habitat resources. The changes in the littoral benthic community in Lake Simcoe likely have wide-ranging implications to higher trophic levels and the cycling of energy in the lake. In addition to impacting nutrient cycling and the benthic invertebrate communities of littoral zones, dreissenid mussels can have large effects on food webs and energy cycling. In chapter 5 I used stable isotope analysis of pre- and post-dreissenid components of the nearshore food web of Lake Simcoe, Ontario to determine how dreissenids affected food sources and energy flow in the littoral zone of Lake Simcoe. Results suggest that the post-dreissenid food web relies about equally on two energy sources: dreissenid biodeposits (redirected pelagic primary production) and littoral benthic primary producers. Although the relative importance of pelagic and benthic primary production to benthic organisms has not changed much following dreissenid establishment, the absolute importance of both increased considerably in the post-dreissenid littoral zone: the large increase in invertebrate biomass that followed dreissenid establishment means that the amount of both pelagic and benthic primary production needed to sustain post-dreissenid organisms had to increase considerably. The results of this chapter suggest that dreissenids increase the availability to food to littoral organisms by redirecting pelagic primary production to the benthos and by stimulating littoral benthic primary production. The impacts of dreissenids on littoral benthic organisms probably have large effects on littoral and pelagic fish communities of lakes. Dreissenid mussels translocate biological production to the benthos by stimulating benthic primary production through nutrient excretion and increases in water clarity, by increasing habitat availability for benthic organisms and by biodepositing pelagic material that becomes available to benthic organisms and the fish that feed on them. I argue that hydrodynamic factors are important in controlling the strength of the dreissenid-mediated pelagic-littoral connection in lakes. Because hydrodynamics relate to lake size, a relationship between lake size and the ability of dreissenids to translocate production the littoral zone can be postulated, where dreissenid effects are maximal in intermediate-sized lakes.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2002. / Bibliography: leaves 78-89.

Marine bioinvasion has been increasing exponentially due to intensive human activities. To manage the threats posed by marine bioinvasion, it is important to understand the key factors for invasion success. Stress tolerance is thought to play an important role in the invasion process, however, little is known of the nature of this tolerance, particularly whether stress tolerance is species- or population-specific. To determine this, laboratory experiments were conducted to investigate differential tolerance of populations of a cosmopolitan marine invasive mussel, Perna viridis, and whether stress tolerance can be altered through pre-selection of individuals. Comparisons were firstly made between two local P. viridis populations from the intertidal and subtidal zones to test if stress tolerance was population-specific. Individuals from the intertidal population had higher baseline heart rates, lower critical salinity values, Scrit, for cardiac activity and longer survival duration at low salinity than individuals from the subtidal population, supporting the presence of population level differences in stress tolerance. Mortality of P. viridis was also compared at sites in both their introduced range (Hong Kong) and native range (Indonesia) which revealed that individuals from the Hong Kong population were more stress tolerant than their native counterparts in Indonesia. Using the same experimental approach, the effect of pre-selection to stress (a prior pre-selection to hypoxia and a subsequent exposure to heat) showed that stress tolerance can be enhanced by pre-selection. The second part of this study investigated whether invasive mussels, Brachidontes variabilis and P. virdis, were more stress tolerant than the native, non-invasive, Septifer virgatus. Laboratory experiments compared the Scrit as well as critical temperature, Hcrit for cardiac activity. Mortality and byssus thread production were also measured in a factorial design with different combinations of temperature (16, 24 and 32 °C) and salinity (8, 16, 32 p.s.u) for 15 days. The two invasive mussels had a higher Hcrit and lower Scrit, which indicated a higher stress tolerance, than the non-invasive mussel. The mortality rate of P. viridis was faster than in B. variabilis and S. virgatus at 8 p.s.u. whereas S. virgatus was the only mussel that exhibited mortality in the medium hyposalinity (16 p.s.u.) and showed much slower byssus attachment than the invasive mussels, suggesting that the invasive mussels are more stress tolerant to heat and medium hyposalinity than the non-invasive species. Overall, stress tolerance proved to be higher in the invasive than non-invasive species and was also population-specific with higher stress tolerance in the invasive population, which may be attributed to the selection of more tolerant individuals during the invasion process. Such a process may occur when founding individuals successfully pass through an invasion process, which may increase the overall mean population-level of stress tolerance and explain why invasive species are generally more stress tolerant than native species. Thus stress tolerance plays an important role in invasion success and invasive species with high stress tolerance may be favoured by climate change, including ocean warming and increased precipitation, which may extend their current distribution range. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy