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Halophilic bacteria and archaea as food sources for the brine shrimp Artemia sp.

(2020)
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Abstract
The stagnation of capture fisheries, combined with the increasing demand of seafood by the world’s growing population, urges for the sustained high growth of aquaculture as a keystone in global food security. Yet, the production of sufficient live food for the larviculture stage is still a bottleneck for aquaculture expansion and diversification. As a substitute for the natural food, the larvae (nauplii) of the brine shrimp Artemia are used as a universal live food source for many aquaculture organisms thanks to its convenience, flexibility in use, and nutritional value. In order to match the increasing demand for Artemia cysts, it is imperative to optimize the management of both its natural sources at salt lakes and its farming in solar salt ponds. Management of Artemia populations has until now mainly focused on the importance of microalgae to nutritionally sustain high Artemia densities. However, given the significance of halophilic Bacteria and Archaea in the microbial biomass composition of these hypersaline environments (see Chapter 1), it becomes clear that a thorough understanding of how Artemia takes dietary advantage of the various types of microbiota present in its environment is essential for its optimal management. The present thesis aimed therefore to investigate the ability of Artemia to use halophilic Bacteria and Archaea as food sources and to evaluate the potential of these microbes to be valorised as complements to microalgae biomass diets in Artemia culture. Firstly, the capacity of Artemia nauplii to survive and grow on mono-diets consisting exclusively of halophilic bacteria typical for the hypersaline environments where it occurs was evaluated in gnotobiotic culture conditions (Chapter 2). The utilization of gnotobiotic Artemia was important in this study since in this model culture system any possible alternative food sources that are naturally present in conventional culture systems are eliminated, and therefore more conclusive results can be obtained. The overall positive performance of Artemia nauplii when fed with the tested halophilic Bacteria demonstrated that they are a viable food source for Artemia and suggests that these microorganisms may indeed be part of its diet in saline environments. The next step of this research investigated for the first time the existence of trophic interactions between Artemia and the domain Archaea, more specifically with the halophilic Archaea (haloarchaea), which are also present in the hypersaline environment where Artemia occur. Firstly, a haloarchaea stable isotopes labelling procedure, using the isotopes 13C and 15N was tested in order to enable the application of isotopic tracers in the investigation of haloarchaea consumption by Artemia or any other aquatic metazoan (Chapter 3). Among the three 13C enriched carbon sources and two 15N enriched ammonium salts tested as potential labels to enrich cells of haloarchaea when supplemented to the culture medium, 13C-glycerol was the most effective 13C label, while both ammonium salts, (15NH4)2SO4 and 15NH4Cl were effective 15N labels. The haloarchaea stable isotopes labelling procedure using glycerol as 13C enriched carbon source and subsequent haloarchaea biomass assimilation experiments using 13C isotope as tracer were then applied in combination with gnotobiotic culture tests to evaluate the ability of Artemia nauplii to assimilate nutrients from mono-diets consisting of haloarchaea biomass (Chapter 4). The study demonstrated the ability of Artemia to assimilate nutrients from the tested mono-diets, providing clear indications that in addition to the consumption of phytoplankton and bacteria, haloarchaea may also be part of the natural Artemia diet. In order to evaluate the potential of these halophilic prokaryotes to be valorised as complements to microalgae diets in the culture of Artemia during its full life cycle, a conventional (non-gnotobiotic) 16-days laboratory culture experiment was conducted where ongrowing Artemia was fed a sub-optimal microalgae supply, supplemented with biomass of these microbes (Chapter 5). Each combination of algae and tested halophilic Bacteria/Archaea was offered in a proportion of 25% microalgae / 75% Bacteria/Archaea on an ash free dry weight basis, while the total amount of food supplied, which was based on a reference algae mono-diet, was kept constant. Mono-diets consisting of 100% microalgae and only 25% microalgae were used as controls. Although resulting in lower survival, the tested microbial biomass was able to partially or completely compensate for the sub-optimal microalgae supply in terms of growth and final Artemia biomass. Furthermore, a significantly higher proportion of mature animals was obtained when using the mixed diets. The study demonstrated therefore that halophilic Bacteria and Archaea are not only a source of nutrients to early Artemia stages, but continue to be a source of nutrients for Artemia throughout its life cycle and enable it to attain and even advance sexual maturation. This is of special relevance for management strategies of Artemia populations in solar salt ponds, since it indicates that the stimulation of heterotrophic microbes as an additional food source may have a direct impact in terms of culture productivity. Since halophilic microbiota flourish in extreme environmental conditions, they have evolved to produce enzymes and metabolites allowing to develop biological activities while assuring cellular and functional integrity, in conditions in which their marine counterparts could not be functional. Several specific compounds, namely poly-ß-hydroxybutyrate (PHB), that are known to convey protection against stressors in aquatic organisms, such as in Artemia, are believed to be accumulated in larger amounts by these organisms. Therefore, this research also identified the strains with high PHB accumulation capability among the halophilic Bacteria and Archaea that had shown dietary value for Artemia in previous chapters, by testing different culture conditions previously described as optimal for PHB stimulation in halophilic microorganisms. The protective effects of these PHB accumulating halophilic strains to gnotobiotic Artemia were then assessed during a pathogenic Vibrio campbellii challenge (Chapter 6). The obtained results indicated the existence of promising protective effects of PHB accumulating halophilic Bacteria and Archaea for Artemia. This not only demonstrates that halophilic Bacteria and Archaea biomass consumption may benefit Artemia by increasing their robustness to potential pathogens, but also opens new perspectives regarding the potential application of Bacteria and Archaea originating from hypersaline environments as probiotics in the aquaculture industry, specifically for the use of PHB as an alternative to antibiotics in the culture of farmed animals (e.g. fish, crustaceans). Finally, the General Discussion (Chapter 7) aimed to review and integrate these results in a larger scientific framework. The referred section also highlights the implications of this study and suggests potential perspectives to the future research. In summary, our results significantly expanded our understanding of the relationships between Artemia and its naturally associated prokaryotic microbiota at hypersaline environments, not only demonstrating the existence of trophic interactions between Artemia and halophilic Bacteria and Archaea, showing that they may be valorised as dietary complements to microalgae in Artemia culture, but also indicating that these interactions may have a direct effect on Artemia fitness.

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Citation

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MLA
Alfama Lopes Dos Santos, Ruy Miguel. Halophilic Bacteria and Archaea as Food Sources for the Brine Shrimp Artemia Sp. Universiteit Gent. Faculteit Bio-ingenieurswetenschappen, 2020.
APA
Alfama Lopes Dos Santos, R. M. (2020). Halophilic bacteria and archaea as food sources for the brine shrimp Artemia sp. Universiteit Gent. Faculteit Bio-ingenieurswetenschappen.
Chicago author-date
Alfama Lopes Dos Santos, Ruy Miguel. 2020. “Halophilic Bacteria and Archaea as Food Sources for the Brine Shrimp Artemia Sp.” Universiteit Gent. Faculteit Bio-ingenieurswetenschappen.
Chicago author-date (all authors)
Alfama Lopes Dos Santos, Ruy Miguel. 2020. “Halophilic Bacteria and Archaea as Food Sources for the Brine Shrimp Artemia Sp.” Universiteit Gent. Faculteit Bio-ingenieurswetenschappen.
Vancouver
1.
Alfama Lopes Dos Santos RM. Halophilic bacteria and archaea as food sources for the brine shrimp Artemia sp. Universiteit Gent. Faculteit Bio-ingenieurswetenschappen; 2020.
IEEE
[1]
R. M. Alfama Lopes Dos Santos, “Halophilic bacteria and archaea as food sources for the brine shrimp Artemia sp.,” Universiteit Gent. Faculteit Bio-ingenieurswetenschappen, 2020.
@phdthesis{8665569,
  abstract     = {{The stagnation of capture fisheries, combined with the increasing demand of seafood by the world’s growing population, urges for the sustained high growth of aquaculture as a keystone in global food security. Yet, the production of sufficient live food for the larviculture stage is still a bottleneck for aquaculture expansion and diversification. As a substitute for the natural food, the larvae (nauplii) of the brine shrimp Artemia are used as a universal live food source for many aquaculture organisms thanks to its convenience, flexibility in use, and nutritional value. In order to match the increasing demand for Artemia cysts, it is imperative to optimize the management of both its natural sources at salt lakes and its farming in solar salt ponds. Management of Artemia populations has until now mainly focused on the importance of microalgae to nutritionally sustain high Artemia densities. However, given the significance of halophilic Bacteria and Archaea in the microbial biomass composition of these hypersaline environments (see Chapter 1), it becomes clear that a thorough understanding of how Artemia takes dietary advantage of the various types of microbiota present in its environment is essential for its optimal management. The present thesis aimed therefore to investigate the ability of Artemia to use halophilic Bacteria and Archaea as food sources and to evaluate the potential of these microbes to be valorised as complements to microalgae biomass diets in Artemia culture. 
Firstly, the capacity of Artemia nauplii to survive and grow on mono-diets consisting exclusively of halophilic bacteria typical for the hypersaline environments where it occurs was evaluated in gnotobiotic culture conditions (Chapter 2). The utilization of gnotobiotic Artemia was important in this study since in this model culture system any possible alternative food sources that are naturally present in conventional culture systems are eliminated, and therefore more conclusive results can be obtained. The overall positive performance of Artemia nauplii when fed with the tested halophilic Bacteria demonstrated that they are a viable food source for Artemia and suggests that these microorganisms may indeed be part of its diet in saline environments. 
The next step of this research investigated for the first time the existence of trophic interactions between Artemia and the domain Archaea, more specifically with the halophilic Archaea (haloarchaea), which are also present in the hypersaline environment where Artemia occur. Firstly, a haloarchaea stable isotopes labelling procedure, using the isotopes 13C and 15N was tested in order to enable the application of isotopic tracers in the investigation of haloarchaea consumption by Artemia or any other aquatic metazoan (Chapter 3). Among the three 13C enriched carbon sources and two 15N enriched ammonium salts tested as potential labels to enrich cells of haloarchaea when supplemented to the culture medium, 13C-glycerol was the most effective 13C label, while both ammonium salts, (15NH4)2SO4 and 15NH4Cl were effective 15N labels.
 The haloarchaea stable isotopes labelling procedure using glycerol as 13C enriched carbon source and subsequent haloarchaea biomass assimilation experiments using 13C isotope as tracer were then applied in combination with gnotobiotic culture tests to evaluate the ability of Artemia nauplii to assimilate nutrients from mono-diets consisting of haloarchaea biomass (Chapter 4). The study demonstrated the ability of Artemia to assimilate nutrients from the tested mono-diets, providing clear indications that in addition to the consumption of phytoplankton and bacteria, haloarchaea may also be part of the natural Artemia diet.
In order to evaluate the potential of these halophilic prokaryotes to be valorised as complements to microalgae diets in the culture of Artemia during its full life cycle, a conventional (non-gnotobiotic) 16-days laboratory culture experiment was conducted where ongrowing Artemia was fed a sub-optimal microalgae supply, supplemented with biomass of these microbes (Chapter 5). Each combination of algae and tested halophilic Bacteria/Archaea was offered in a proportion of 25% microalgae / 75% Bacteria/Archaea on an ash free dry weight basis, while the total amount of food supplied, which was based on a reference algae mono-diet, was kept constant. Mono-diets consisting of 100% microalgae and only 25% microalgae were used as controls. Although resulting in lower survival, the tested microbial biomass was able to partially or completely compensate for the sub-optimal microalgae supply in terms of growth and final Artemia biomass. Furthermore, a significantly higher proportion of mature animals was obtained when using the mixed diets. The study demonstrated therefore that halophilic Bacteria and Archaea are not only a source of nutrients to early Artemia stages, but continue to be a source of nutrients for Artemia throughout its life cycle and enable it to attain and even advance sexual maturation. This is of special relevance for management strategies of Artemia populations in solar salt ponds, since it indicates that the stimulation of heterotrophic microbes as an additional food source may have a direct impact in terms of culture productivity. 
Since halophilic microbiota flourish in extreme environmental conditions, they have evolved to produce enzymes and metabolites allowing to develop biological activities while assuring cellular and functional integrity, in conditions in which their marine counterparts could not be functional. Several specific compounds, namely poly-ß-hydroxybutyrate (PHB), that are known to convey protection against stressors in aquatic organisms, such as in Artemia, are believed to be accumulated in larger amounts by these organisms. Therefore, this research also identified the strains with high PHB accumulation capability among the halophilic Bacteria and Archaea that had shown dietary value for Artemia in previous chapters, by testing different culture conditions previously described as optimal for PHB stimulation in halophilic microorganisms. The protective effects of these PHB accumulating halophilic strains to gnotobiotic Artemia were then assessed during a pathogenic Vibrio campbellii challenge (Chapter 6). The obtained results indicated the existence of promising protective effects of PHB accumulating halophilic Bacteria and Archaea for Artemia. This not only demonstrates that halophilic Bacteria and Archaea biomass consumption may benefit Artemia by increasing their robustness to potential pathogens, but also opens new perspectives regarding the potential application of Bacteria and Archaea originating from hypersaline environments as probiotics in the aquaculture industry, specifically for the use of PHB as an alternative to antibiotics in the culture of farmed animals (e.g. fish, crustaceans). 
Finally, the General Discussion (Chapter 7) aimed to review and integrate these results in a larger scientific framework. The referred section also highlights the implications of this study and suggests potential perspectives to the future research. In summary, our results significantly expanded our understanding of the relationships between Artemia and its naturally associated prokaryotic microbiota at hypersaline environments, not only demonstrating the existence of trophic interactions between Artemia and halophilic Bacteria and Archaea, showing that they may be valorised as dietary complements to microalgae in Artemia culture, but also indicating that these interactions may have a direct effect on Artemia fitness.}},
  author       = {{Alfama Lopes Dos Santos, Ruy Miguel}},
  isbn         = {{9789463573320}},
  language     = {{eng}},
  pages        = {{XV, 169}},
  publisher    = {{Universiteit Gent. Faculteit Bio-ingenieurswetenschappen}},
  school       = {{Ghent University}},
  title        = {{Halophilic bacteria and archaea as food sources for the brine shrimp Artemia sp.}},
  year         = {{2020}},
}