The importance of aquaculture
Aquaculture encompasses the cultivation of aquatic animals and plants in various water types and is the fastest growing food-producing sector globally, as noted by Subasinghe et al (2009) It contributes significantly to the global protein intake, with fish and fishery products accounting for 16.6% of animal protein consumption (FAO 2014a) While capture fisheries production has remained relatively stable, aquaculture's output has risen steadily, reaching 73% of capture fisheries production by 2012 and matching it by 2015 This growth positions aquaculture as a vital solution to meet future food demands Additionally, it plays a crucial role in generating employment and trade, particularly benefiting rural areas facing challenges from population pressure and environmental degradation (Finegold 2009; Halwart 2005).
Figure 1.1 The global capture and aquaculture production (FAO 2014)
The immediate goals of the industrial aquaculture
Sustainable development is the most important target of industrial aquaculture in coming years To accomplish that there are some priority needs which have been suggested by Sorgeloos (2013):
(i) Domestication: complete independence from natural stocks such as wild breeders or seed through domestication of aquatic species
Improved seed production can be achieved through the development of new hatchery practices, such as microbial steering, and the application of innovative products like specialized substrates for specific bacteria or signal molecules that disrupt virulence triggers This approach not only enhances the efficiency of seed production but also reduces costs, contributing to more sustainable aquaculture practices.
(iii) Species selection: more selective in identifying suitable species for mass markets and niche species catering to local markets where value-added products might be in good demand
(iv) Selective breeding: development of more efficient stock through selective breeding, especially genomic sequence comparisons with model species can help to identify best breeding goals
Bacteria play a crucial role in aquaculture, influencing both sustainable production and microbial management As water serves as a prime environment for microbial growth, understanding the functions of both beneficial and harmful bacteria in aquaculture systems is essential Increased research focus on these microbial dynamics is necessary to enhance sustainability in aquaculture practices.
(vi) Health control: more basic work using molecular tools should improve knowledge of activation, good functioning and disruption of the animal’s immune systems, especially in invertebrates, crustaceans and mollusks
(vii) Ecological aquaculture: more integrated farming of terrestrial and aquatic plants and animals for sensor-controlled nutrient dosing and heat, energy and water recovery
Marine aquaculture plays a crucial role in utilizing the vast resources of our oceans and seas, which account for 70% of the planet's aquatic resources Despite this, only 50% of global aquatic products are sourced from marine environments, with nearly half of that being aquatic plants This highlights the need for greater focus on sustainable practices in marine aquaculture to enhance production and protect marine ecosystems.
(ix) Replacements for fishmeal and fish oils: full independence from natural fish stocks for lipid and protein ingredients in production of aquatic feeds
(x) Stock enhancement programs: more attention for integration of restocking activities with fisheries management in freshwater and marine environments.
Importance of crustacean aquaculture
Crustaceans are a highly valued aquaculture product with significant global demand, as evidenced by their production growth from approximately 3 million tonnes in 2003 to 6.45 million tonnes in 2012 (FAO 2014) Recent statistics indicate that crustacean aquaculture accounts for about 7% of the total global aquaculture production (FAO 2014).
2012 (Fig 1.2) Despite this modest quantity, it represented a value of about 30.9 billion US dollars, which is 21.4% of the total value of the world aquaculture production
Figure 1.2 World aquaculture production of major species groups in 2012 by quantity and value: crustaceans are indicated with an arrow (FAO 2014)
Giant freshwater prawn (Macrobrachium rosenbergii)
The giant freshwater prawn, Macrobrachium rosenbergii (De Man 1987), is distributed in
M rosenbergii, a native species found in Southeast Asia, South Pacific countries, Northern Oceania, and Western Pacific islands, typically inhabits freshwater environments such as rivers, lakes, ditches, canals, and coastal pools Since its universal acceptance in 1959, it has been recognized under various names.
The phylogenetic characterization of M rosenbergii is:
English name Giant freshwater prawn
The morphology of adult M rosenbergii is described by Nandlal and Pickering (2006) The main morphological characteristics are shown in Fig 1.3:
Figure 1.3 External anatomy of Macrobrachium rosenbergii (Nandlal and Pickering 2006)
Tropical freshwater environments, connected to adjacent brackish water bodies are the living environment of M rosenbergii Brackish water is the habitat of its larvae (Sandifer et al
In 1975, it was observed that gravid females of M rosenbergii migrate downstream into estuaries to spawn, where their eggs hatch into free-swimming larvae after 18 to 23 days at 28°C These newly hatched larvae swim upside down and tail first, seeking brackish water with optimal salinity levels of 9 to 19 g/L for better survival Over a period of 15 to 40 days, the larvae undergo eleven metamorphic stages before reaching the postlarvae stage, influenced by factors such as temperature and food quality During this time, their primary diet consists of zooplankton and small aquatic invertebrates Once in the postlarval stage, they adapt to a range of salinities and begin migrating upstream toward freshwater habitats.
Figure 1.4 The life cycle of Macrobrachium rosenbergii (New and Valenti 2000)
1.4.2 The status of Macrobrachium rosenbergii culture
M rosenbergii is not only a high value food source but also has a high economical value as an export product Furthermore, M rosenbergii farming does not need a high investment nor is it as technically demanding or capital intensive as the farming of black tiger or whiteleg shrimp It is more environmentally sustainable due to lower culture density (Nandlal and Pickering 2006) Nowadays, the farming of M rosenbergii is performed in many countries;
China, India, Vietnam, Thailand, Bangladesh and Taiwan are the main producers of M rosenbergii (New 2005) According to the FAO aquaculture production statistics for the year
2012, the global contribution of M rosenbergii to aquaculture production has reached over
Figure 1.5 Global production of Macrobrachium rosenbergii from 1970 to 2012 (FAO
The commercial aquaculture of prawn relies on postlarvae sourced from both the wild and hatcheries; however, the current availability of wild seed is inadequate to satisfy the growing demands of the prawn farming industry, with inconsistencies in size also being a concern To ensure the sustainability of farming M rosenbergii, it is crucial to implement reliable hatching techniques and effective management practices for the artificial propagation of prawn seed.
Broodstock: Often only berried female prawns are kept in hatcheries until their eggs hatch
The term 'berried' or 'ovigerous' refers to adult female aquatic species that carry eggs beneath their tails, which can be sourced from farm ponds or natural bodies of water such as rivers, canals, and lakes Research indicates that the fecundity, egg hatchability, and overall quality of larvae can be significantly enhanced when broodstock are provided with a diet rich in highly unsaturated fatty acids (HUFA), specifically 18:2n-6 and n-3, at levels of 13 and 15 mg/g dry weight of food, respectively.
Egg incubation involves selecting ovigerous female prawns from wild or farm ponds, which are then placed in communal or separate tanks containing brackish water until they hatch The techniques employed for this process vary based on geographical location and the scale of the hatchery.
Larval rearing involves nurturing the first larval stage, which measures less than 2 mm and is collected post-hatching This stage thrives at an optimal temperature of 28–31 °C and a salinity of 12 g/L, with the larval cycle typically lasting 25–30 days Hatcheries primarily utilize two culture methods: flow-through systems, where rearing water is continuously renewed, and recirculating systems, which employ physical and biological filters to reduce water usage The primary diet for larval prawns consists of newly hatched Artemia nauplii.
In the hatchery phase, a diverse range of larval feeds is utilized, including Artemia nauplii, fish eggs, squid flesh, frozen and flaked adult Artemia, fish flesh, egg custard, worms, and commercial feeds (Nhan 2009) Research indicates that supplementing or enriching the feed with (n-3) highly unsaturated fatty acids (HUFAs) can significantly enhance the growth of M rosenbergii (Romdhane et al 1995; Alam et al 1995).
In the grow-out phase, postlarvae of M rosenbergii, measuring 15-20 mm and weighing 0.015-0.020 g, are nurtured in tanks or ponds until they reach a size of 3-5 g before being transferred to grow-out ponds at a density of 5-8 postlarvae/m² for monoculture These prawns benefit from natural food sources like small snails, shellfish, worms, grains, nuts, and fruit, alongside supplementary formulated feeds that typically contain 30-35% protein, 2-10% fat, and 4-12% fiber, available in various forms such as powder, meal, crumble, or pellets to cater to different growth stages The pond grow-out phase lasts approximately 4-6 months, and M rosenbergii can also be raised in polyculture with other aquatic species.
The increased production of M rosenbergii in recent decades has been accompanied by frequent disease outbreaks in both grow-out ponds and hatcheries, significantly impacting the quantity and quality of seed produced (New and Valenti 2000; Shailender et al 2012) Various pathogens, particularly opportunistic ones like Vibrio spp., have been responsible for these diseases, which have adversely affected the commercial culture of M rosenbergii (Tongguthai 1995; Kennedy et al 2006).
Research indicates that high mortality rates in hatchery systems can be attributed to various pathogens (Jayaprakash et al., 2006; Sharshar and Azab, 2008; Skjermo and Vadstein, 1999) According to Shailender et al (2012), the primary pathways for these pathogens to infiltrate hatcheries include contaminated feed, broodstock, instruments, water, and poor hygiene practices among workers.
Table 1.1 outlines common diseases and control measures in the farming and hatcheries of M rosenbergii While antibiotics and pharmaceuticals are sometimes utilized for treatment, their presence in this table does not indicate a recommendation from the FAO (FAO 2002).
Disease Agent Type Syndrome Measures
Infected tissue becomes opaque, with progressive necrosis; affects juveniles
Baculovirus Virus White spot; affects juveniles IH
Unnamed viral disease Nodavirus Virus Whitish tail; affects larvae IH
Black spot; brown spot; shell disease
Melanised lesions; affects all life stages, but more frequently observed in juveniles and adults
Similar to black spot but only affects larvae, especially stages IV and V
IH; nifurpurinol; erythromycin; penicillin- streptomycin; chloramphenicol
Improved husbandry (IH) involves effective management practices that prioritize hygiene, proper breeding care, and appropriate feeding activities Key elements include avoiding overstocking and overfeeding, as well as ensuring the sanitary disposal of dead animals and leftover food throughout the culture period.
Disease Agent Type Syndrome Measures
Luminescent larval syndrome Vibrio harveyi Bacterium Moribund and dead larvae luminescent
White postlarval disease Rickettsia Bacterium
White larvae, especially stages IV and V
IH; oxytetracyline; furazolidone; lime, prior to stocking
Unnamed fungal infection Lagenidium Fungus
Extensive mythelial network visible through exoskeleton of larvae
Unnamed fungal infection (often associated with
Fusarium solani Fungus Secondary infection; affects adults IH
Yellowish grayish or bluish muscle tissue in juvenile
External parasites that inhibit swimming, feeding and moulting; affect all life stages
IH; formalin; merthiolate; copper-based algicides
Disease Agent Type Syndrome Measures
Whitish color in striated tissue of tail and appendages; when advanced, necrotic areas may become reddish; affects all life stages
Lethargy; spiraling swimming; reduced feeding and growth; bluish-grey body color; affects larvae, especially stages VI and VII
Disease), sometimes known as MDS
Unknown but probably multiple causes, including nutritional deficiency
Localised deformities; failure to complete moulting; affects late larval stages; also seen in postlarvae, juvenile and adults
The brine shrimp Artemia
1.5.1 Biology and ecology of Artemia
The brine shrimp Artemia, a small crustacean zooplankton, thrives in hypersaline environments with salt concentrations reaching up to 250 g/L This genus includes both sexual species and parthenogenetic lineages, allowing Artemia to reproduce in two distinct ways Under favorable conditions, fertilized eggs develop into free-swimming nauplii through ovoviviparous reproduction, while adverse conditions trigger alternative reproductive strategies.
Artemia has the ability to produce dormant embryos of about 200 - 300 àm, cysts covered by a tough brown shell, that are in a state of obligate dormancy called diapause (Lavens and Sorgeloos 1987)
Figure 1.6 Life cycle of Artemia Ovoviviparous offspring production is indicated by the dashed line, while oviparous offspring production is indicated by the full line (Jumalon et al
Cysts reactivate their metabolic functions only after complete dehydration and subsequent rehydration under optimal conditions, such as appropriate salinity, temperature, and aeration During the hydration process, aerobic metabolism initiates critical conversions, including trehalose to glycogen for energy and trehalose to glycerol for hatching support Approximately 8 to 20 hours post-hydration, an embryo develops within a surrounding hatching membrane, and just before the cyst shell ruptures, the free-swimming Artemia nauplius instar I emerges.
The newly hatched nauplius, measuring approximately 400-500 µm in length, does not feed and depends on yolk energy for survival It features a distinctive red nauplius eye and possesses three pairs of appendages: the first antennae serve a sensory role, the second antennae are used for locomotion and filter feeding, and the mandibles are responsible for food uptake Around eight hours post-hatching, the instar I nauplius undergoes further development.
15 the instar II (or metanauplius) stage and can start to take up exogenous particles (Campbell et al 1993; Dixon et al 1995; Van Stappen 1996; Sorgeloos et al 2001)
Figure 1.7 Artemia nauplius with three pairs of appendages
(Source: http://www.optics.rochester.edu/workgroups/cml/opt307/spr10/jonathan/)
Artemia metanauplii take up smaller food particles (e.g algal cells, bacteria and detritus) with a size ranging from 1 to 50 μm from the water phase by a filteration process using the
Artemia nauplii primarily utilize their second antennae for feeding, ingesting particles that are ideally sized between 6.8 and 27.5 μm, with an optimal size of around 16.0 μm (Van Stappen, 1996; Fernández, 2001).
Research indicates that the uptake of particles sized 4 – 10 μm by Artemia nauplii is influenced by both the dose and exposure time Studies on the morphology of feeding appendages in Artemia metanauplii reveal that the inter-setular distance serves as a key indicator for the minimum size of food particles that can be effectively filtered Furthermore, findings by Makridis and Vadstein (1999) demonstrate that the maximum filtration rate of Artemia franciscana metanauplii increases with development, reaching rates of 50 - 60, 254, and 1480.
In a study on metanauplii, the oxygen consumption rates were measured at 2100 μL/ind./h for 2-, 4-, and 7-day-old specimens The inter-setular distances in the antennae and thoracopods were found to be 0.20 ± 0.07 μm, 0.16 ± 0.05 μm, and 0.18 ± 0.04 μm for the respective age groups, indicating that these measurements were consistent across different developmental stages (Makridis and Vadstein 1999).
The instar I and II nauplius stages are most often used as a live food for the culture of larval shrimp and fish in aquaculture (Bengtson et al 1991)
1.5.2 The role of Artemia in aquaculture
1.5.2.1 The supply and demand of cysts
Dehydrated Artemia cysts are mainly harvested from the wild in inland salt lakes with the
The Great Salt Lake in Utah, USA, is the leading producer of Artemia cysts, contributing approximately 2,500 to 3,000 tonnes annually for global aquaculture needs, particularly in the cultivation of shrimp and fish across various water types (FAO 2011; Bengtson et al 1991) As industrial aquaculture expands, the demand for Artemia cysts is expected to surge, often outpacing supply from inland salt lakes To address this growing need, Artemia culture has been established in several countries, including Brazil, Thailand, the Philippines, Vietnam, Pakistan, and India, with notable production in the Vinh Chau and Bac Lieu districts.
Mekong Delta, Vietnam was approximately 30 tonnes in 2012 (Toi 2014)
Artemia is widely recognized in industrial aquaculture as a superior live food source for the larvae of fish and crustaceans during their early developmental stages This versatile organism provides numerous benefits, including a sustainable nutritional profile, suitable size for easy ingestion, and the ability for long-term storage, making it an essential component in hatcheries.
Artemia nauplii, which are non-selective particle feeders, undergo a molt into the second instar stage, allowing for the incorporation of various products into them before they are fed to predator larvae Research by Léger et al (1987) indicates that nauplii enriched with highly unsaturated fatty acids (HUFAs) enhance their nutritional profile, resulting in higher energy content and the inclusion of all essential fatty acids, such as 22:6n-3, which are typically scarce in nauplii from most strains Additionally, enrichment techniques can also introduce other vital nutrients, prophylactics, and therapeutics.
Artemia nauplii serve as an effective carrier for oral vaccination of fish fry, particularly when incubated with Vibrio anguillarum antigens (Campbell et al 1993) Research indicates that feeding Peneaus monodon postlarvae enriched Artemia nauplii enhances growth, disease resistance, and stress tolerance This enrichment can include essential highly unsaturated fatty acids (HUFAs), probionts, and herbal products (Immanuel et al 2007).
Artemia nauplii are not only utilized as live food but also as a protein and lipid source in aquatic animal feed (Anh et al 2009; Dhert et al 1993) Research by Anh et al (2011a) indicates that nursery stage Scylla paramamosain crabs thrive on Artemia biomass, whether live or frozen, collected post-Artemia cyst production, leading to improved survival rates compared to those fed fresh shrimp meat Additionally, Artemia can be cultivated in tanks with rice bran and microalgae Tetraselmis suecica, resulting in a biochemical composition akin to that of wild-caught adult Artemia (Teresita et al 2005) Furthermore, Artemia biomass is particularly beneficial for large larvae of carnivorous species, including lobsters (Shleser and Gallagher 1974), mud crabs (Mann et al 2001), and post-larval giant tiger prawns (Penaeus monodon) (Anh et al 2011b).
The use of Artemia nauplii as live food for fish and shrimp larvae poses certain risks due to the potential introduction of opportunistic pathogens During the incubation of cysts for nauplii production, the cyst shells can become contaminated with bacteria, protozoa, or fungi, with bacterial concentrations exceeding 10^7 CFU/mL in the hatching medium Consequently, feeding Artemia nauplii to larvae can serve as a primary pathway for the transmission of harmful bacteria, such as Vibrio spp.
Research by Torres and Lizárraga-Partida (2001) and Interaminense et al (2014) indicates that the proliferation of opportunistic bacteria is directly related to the density of Artemia nauplii production In controlled laboratory settings, Vibrio campbellii and Vibrio harveyi are commonly utilized as pathogens to study the effects on Artemia nauplii under gnotobiotic conditions (Defoirdt et al 2005; Marques et al 2006).
To control bacterial contamination from Artemia in hatcheries, hypochlorite is commonly used for cyst disinfection, effectively eliminating most commercial Artemia strains (Sorgeloos et al 2001) Despite this, the hatching medium can quickly become recolonized by bacteria during incubation, potentially endangering larvae health if pathogenic bacteria are present (Sorgeloos et al 2001) To address this issue, specialized products like INVE’s Sanocare ACE have been developed to reduce pathogenic bacterial growth in hatching tanks (Delbos and Schwarz 2009) Although the active ingredient in this proprietary product is undisclosed, it is primarily derived from herbal sources.
1.5.2.4 Artemia as a model test organism
Artemia serves as a valuable model organism in aquaculture research for crustaceans, enabling higher throughput in trials compared to target aquaculture species This is due to the smaller scale of experimental setups, which allows for a greater number of replicates and treatments, as well as the rapid production of Artemia nauplii from cysts.
18-24 hours), the short generation time of ca 2-3 weeks for the production of live offspring by adults, and the possibility to work with sterile Artemia nauplii (Marques et al 2004a;
Measures to control diseases in aquaculture
Efforts to manage disease in aquaculture have primarily focused on targeting pathogens within the host, with antibiotics being the most common strategy These antibiotics serve multiple purposes, including therapeutic treatment for sick animals, prophylactic measures to prevent illness, and as feed additives to enhance growth performance However, the frequent prophylactic use of antibiotics, especially at suboptimal doses, has led to the emergence of antibiotic-resistant pathogens and heightened the risk of resistant plasmid transfer to humans and domesticated animals Consequently, researchers worldwide are challenged to develop alternative methods to replace antibiotics in aquaculture.
Controlling diseases in aquaculture begins with environmental management, particularly through the disinfection of water to prevent pathogen invasion Effective disinfection methods, such as the use of lime and hypochlorite, have been traditionally employed to eliminate all living organisms, including potential pathogens, before stocking animals This approach is based on the premise that by removing the causative agents, the risk of disease can be significantly reduced (Summerfelt et al 2009; Cruz-Lacierda and De Le Peña, 1996; Tonguthai, 2000).
Ozone (O3) is a powerful oxidizing agent used as an alternative method for controlling pathogenic bacteria and fungi by destroying their outer membranes Unlike traditional methods like lime and hypochlorite, O3 is typically applied within a recirculation system during animal culture However, its use must be approached with caution, as it can eliminate not only harmful bacteria but also beneficial ones essential for animal growth Additionally, the implementation of O3 involves significant investment costs.
2001) while it may also be toxic for the animals (Tango and Gagnon 2003)
Ultraviolet (UV) irradiation is used as an alternative to O3 to kill pathogens in the water of the recirculation systems by denaturing the DNA of microorganisms (Summerfelt 2003;
The effectiveness of UV irradiation in water treatment is influenced by water turbidity, and like ozone treatment, it not only eliminates harmful pathogens but also affects beneficial bacteria.
Immunostimulants are natural compounds that enhance the immune response of cultured animals, increasing their resistance to diseases (Bricknell and Dalmo, 2005) These substances can include live or killed bacteria, glucans, peptidoglycans, and lipopolysaccharides (Smith et al., 2003) Both fish and crustaceans can benefit from immunostimulation, with fish experiencing activated lymphocytes and enhanced phagocytic activity (Sakai, 1999) In crustaceans, immunostimulants lead to increased haemocyte activity, which is crucial for combating infections through mechanisms like phagocytosis and the release of antimicrobial substances (Smith et al., 2003) Studies have demonstrated that immunostimulants provide protection to crustaceans such as Penaeus indicus and Penaeus monodon against luminescent vibriosis and improve the immunity of Penaeus monodon against white spot syndrome virus (Alabi et al., 1999; Thanardkit et al., 2002; Chang et al., 2003; Soltanian et al., 2007).
Vaccination serves as an effective alternative to immunostimulation for preventing diseases like vibriosis and pasteurelosis in aquaculture (Press and Lillehaug 1995) Its primary aim is to stimulate long-term specific immunity against targeted pathogens through the use of specific antigens (Ellis 1988) However, vaccinating crustacean species poses challenges compared to fish, as invertebrates are generally believed to lack immunological memory (Rowley and Pope 2012) Despite this, various studies have demonstrated that vaccination can enhance shrimp survival and provide disease prevention.
21 pathogenic growth in experimental “vaccination” trials with white spot syndrome virus or with vibrios (Mavichak et al 2010; Chotigeat et al 2007; Powell et al 2011)
Another example of a sustainable disease control strategy is quorum sensing (QS) regulation
Quorum sensing (QS) is a bacterial communication process that enables the coordination of gene expression in response to small signaling molecules known as acylated homoserine lactones or autoinducers In aquaculture, disrupting QS is being explored as a novel anti-infective strategy, currently in the experimental stage Research efforts aim to control infections through various methods, including inhibiting signal synthesis, utilizing QS antagonists, chemically inactivating QS signals with oxidized halogen antimicrobials, degrading signal molecules with bacterial lactonases and acylases, and applying QS agonists Many studies on QS biocontrol have concentrated on crustacean species.
Artemia (Defoirdt et al 2006a) and M rosenbergii larvae (Nhan et al 2010a; Pande et al
Recent studies in aquaculture disease control emphasize the role of probiotics and prebiotics Verschuere et al (2000b) redefine probiotics for aquaculture as "live microbial adjuncts" that positively influence the host by altering microbial communities, improving feed utilization and nutritional value, enhancing disease resistance, and improving environmental quality Probiotics are known to regulate intestinal microbiota growth, inhibit harmful bacteria, and strengthen natural defense mechanisms (Giorgio et al 2010) Their effectiveness against pathogens may involve several mechanisms, including the production of inhibitory compounds, competition for nutrients, competition for adhesion sites in the gastrointestinal tract, and enhancement of host immune responses.
Microalgae, such as Tetraselmis sp., and various yeasts, including Debaryomyces sp and Phaffia sp., play a crucial role in enhancing immune responses and producing essential nutrients like vitamins and fatty acids Additionally, they contribute significantly to the digestive process through their enzymatic activities (Verschuere et al 2000b; Vine et al 2006).
Saccharomyces species, along with various Gram-positive bacteria such as Bacillus, Lactococcus, Micrococcus, Carnobacterium, Enterococcus, Lactobacillus, Streptococcus, and Weissella, play significant roles in fermentation processes Additionally, Gram-negative bacteria including Aeromonas, Alteromonas, and Photorhodobacterium contribute to diverse ecological functions.
Pseudomonas sp and Vibrio sp.) have been termed as probiotics (Gatesoupe 1999; He et al
Numerous studies have investigated various probiotic bacteria for aquaculture species, leading to the availability of commercial products in liquid or powder forms, including spore and freeze-dried options Advances in production technologies aim to enhance the functionality of probiotics and improve aquaculture performance Probiotics can be administered to aquatic hosts through several methods: incorporating them into live food, bathing, adding them to culture water, or mixing them into artificial diets.
Probiotic studies on crustacean species must evaluate not only disease resistance and growth performance but also the persistence of the probiotic in the host's intestinal tract This persistence is crucial as it influences the longevity of the treatment and ultimately impacts the cost-effectiveness of probiotic application.
Prebiotics, defined by Ringứ et al (2010) as non-digestible components, are metabolized by beneficial bacteria while inhibiting harmful pathogens, thereby enhancing intestinal health Their primary function is to stimulate health-promoting microorganisms in the gastrointestinal tract, which helps reduce intestinal pathogens and improve the production of beneficial bacterial metabolites (Manning and Gibson 2004) Additionally, prebiotics play a role in boosting the innate immune system (Song et al 2014) These carbohydrates can be categorized into oligosaccharides or polysaccharides based on their molecular size A crucial aspect of their function is the acidification of the colonic environment, which promotes the production of short-chain fatty acids (SCFAs) through the fermentation of prebiotic compounds, contributing positively to gut health (Bongers and Van den).
Heuvel 2007) Some of the most common prebiotics that have been investigated since their introduction in aquaculture of crustaceans are listed in Table 1.2
Table 1.2 Examples of biocontrol measures against crustacean disease in aquaculture (after De Schryver 2010a)
Crustacean species Antagonist/active compound Disease Probiotic/prebiotic effect Reference
Antagonistic activity of probiotics against pathogens:
Bacillus subtilis UTM126 Bacillus subtillis
Pediococcus pentosaceus and Staphylococcus hemolyticus
Increase in survival up to 80 – 100%
Growth and survival of shrimp
Antagonistic effect in in vitro assay against Vibrio sp isolated from Penaeus monodon Protection against disease Increase in survival
Improvements in water quality, growth, survival, SGR, FCR and other immune parameters
Decrease in the prevalence of WSSV
Mehran and Masoumeh (2012) Rahiman et al (2010)
Crustacean species Antagonist/active compound Disease Probiotic/prebiotic effect Reference
Antagonistic activity of probiotics against pathogens:
Bacillus subtilis and Bacillus megaterium
(Short-chain) fructooligosaccharides Mannanoligosaccharides Isomaltooligosaccharides
Increase in survival up to 19%
Increased growth, feed conversion and survival
Improved disease resistance by enhancing immunity and modulate microbiota in the gut
Zhou et al (2007) Genc et al (2007)
1.6.5 Alcaligenes spp and Bacillus spp as probiotics
Alcaligenes spp are ubiquitous, non-fermentative, Gram-negative rods found in various environments, including marine and fresh water, soil, and sewage Although occasionally isolated from diseased tissues, their role as pathogens in humans, animals, and plants remains poorly understood Alcaligenes eutrophus, also known as Ralstonia eutropha or Cupriavidus metallidurans, is a non-spore-forming bacterium native to soils that can utilize diverse carbon sources and accumulate polyhydroxyalkanoates (PHAs) Initially explored as a source of single-cell protein in the 1970s, its high accumulation of polyhydroxybutyrate (PHB) led to decreased interest in this application, yet it became a leading organism for PHB production due to its ease of cultivation and efficiency in synthesizing PHB in simple media Furthermore, Alcaligenes spp exhibit metal resistance, containing genes for heavy metal resistance on megaplasmids, making them significant in aquaculture research.
Alcaligenes eutrophus combined to an algal diet was shown to increase the survival of blue mussel (Mytilus edulis) larvae (Hung et al 2015)
Bacillus species are rod-shaped, endospore-forming, Gram-positive bacteria that thrive in various natural environments due to their diverse physiological capabilities Many Bacillus species are non-pathogenic to mammals, including humans, and are significant in commercial applications for producing a variety of secondary metabolites such as antibiotics, bio-insecticides, biosurfactants, and enzymes In aquaculture, for instance, poly-β-hydroxybutyrate-hydroxyvalerate (PHB-HV) derived from Bacillus thuringiensis B.t.A102 has been utilized as a potential immunostimulant to enhance the immune response in Oreochromis mossambicus when added to their feed.
(Suguna et al 2014) Several commercial bacilli probiotics have been or are being used in aquaculture such as Biostart ® (Microbial Solutions, Johannesburg, South Africa and
Advanced Microbial Systems, USA), consisting of a mixture of B megaterium, B licheniformis, Paenibacillus polymyxa and two strains of B subtilis (Verschuere et al
2000b), Promarine ® (Sino-Aqua company Kaohsiung, Taiwan), containing 4 strains of B subtilis (Urdaci and Pinchuk 2004), and Sanolife probiotics (INVE Aquaculture NV,
Belgium) containing a mixture of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus Recently, it has been reported that in shrimp farms (Penaeus monodon) in Asia and
Poly-β-hydroxybutyrate as antimicrobial agent in aquaculture
Disease outbreaks significantly hinder the growth of the aquaculture industry, leading to substantial losses in the intensive farming of finfish, mollusks, lobsters, and shrimp, primarily due to luminescent vibrios Traditional methods, such as antibiotic treatment, have been commonly employed to combat these issues.
The use of antibiotics in aquaculture for bacterial population control has led to the emergence of multiple resistances in various pathogens As a result, researchers are exploring alternative strategies, such as short chain fatty acids (SCFAs) and poly-β-hydroxybutyrate (PHB), for effective disease management PHB, in particular, shows promise as an antimicrobial agent due to its insolubility in water, which enhances its uptake efficiency compared to volatile SCFAs.
Polyhydroxyalkanoates (PHAs) are biopolymers produced by a diverse range of bacteria, including both Gram-positive and Gram-negative species from over 75 genera, particularly under conditions of nutrient limitation and excess carbon (Reddy et al., 2003; Tian et al., 2009) In times of carbon scarcity, PHAs serve as vital carbon and energy reserves for the cells (Madison and Huisman).
Polyhydroxyalkanoates (PHAs) are significant biopolymers that can accumulate in cells as discrete granules, reaching up to 90% of the cell's dry weight (Anderson and Dawes, 1990) These polymers are primarily linear, head-to-tail polyesters made up of β-hydroxy fatty acid monomers (Madison and Huisman, 1999) Among them, poly-β-hydroxybutyrate (PHB) is the simplest and most prevalent type, highlighting the importance of PHAs in biotechnology and materials science (Freier et al.).
2002) PHB is the most extensively characterized polymer of all PHAs (Lee 1996)
Figure 1.8 Structural formula of poly-β-hydroxybutyrate
The biosynthetic pathway of poly-3-hydroxybutyrate (PHB) involves three key enzymatic reactions facilitated by distinct enzymes The process begins with β-ketothiolase, encoded by the phbA gene, which catalyzes the condensation of two acetyl coenzyme A (acetyl-CoA) molecules into acetoacetyl-CoA Following this, acetoacetyl-CoA reductase plays a crucial role in the subsequent steps of PHB synthesis.
The enzyme encoded by phbB converts acetoacetyl-CoA into 3-hydroxybutyryl-CoA, which is then polymerized by P(3HB) polymerase, encoded by phbC, to produce polyhydroxybutyrate (PHB) from 3-hydroxybutyryl-CoA monomers (Huisman et al 1989; Reddy et al 2003).
The biosynthetic pathway of poly(3-hydroxybutyrate) (P(3HB)) involves a three-step process facilitated by the enzymes β-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), and PHB polymerase (phbC) These enzymes are encoded by the genes of the phbCAB operon, which is transcribed from a promoter located upstream of phbC, ensuring the production of the complete operon.
β-ketothiolase is identified as the key bottleneck enzyme in the PHB biosynthetic pathway, as it is competitively inhibited by high levels of free Coenzyme A (CoASH) during balanced growth conditions, which occur when acetyl-CoA enters the Krebs cycle (Doi et al., 1988) Conversely, under limiting growth conditions, characterized by an excess of carbon sources and insufficient nutrients like nitrogen, PHB production is enhanced In these scenarios, acetyl-CoA concentrations remain elevated while CoASH levels decrease, leading to the activation of β-ketothiolase (Patnaik, 2005) This mechanism underscores the significance of the carbon to nitrogen (C/N) ratio, with an optimal ratio of 20 often recommended for maximizing PHB production (Rathore et al., 2014).
The aerobic process of PHB production consists of two key phases Initially, essential nutrients such as carbon, nitrogen, and oxygen are utilized for biomass growth In the subsequent phase, when nitrogen becomes limited, the process continues with sufficient carbon and oxygen to facilitate PHB production.
The accumulation of polyhydroxybutyrate (PHB) in bacterial cells is influenced by various environmental conditions, including anaerobic, aerobic, and combined aerobic/anaerobic settings Research indicates that while activated sludge can produce PHB during enhanced biological phosphorus removal, the maximum PHB content achieved under anaerobic conditions is only 50% of what is accumulated under aerobic or combined aerobic/anaerobic conditions (Rodgers and Wu, 2009).
1.7.3 The production and cost of polyhydroxyalkanoates
The presence of polyhydroxyalkanoates was first found in Bacillus megaterium in 1925 by
Polyhydroxyalkanoates (PHAs) serve as intracellular reserves for carbon and energy in the genus Bacillus (Lemoigne, Dawes 1988; Sudesh et al 2000) Various bacteria capable of producing PHAs can be isolated from diverse environments, including Bacillus megaterium SW1-2 from activated sewage sludge (Berekaa and Al Thawadi 2012), Ralstonia spp from soils (Bonatto et al 2004), and Bacillus spp from the intestines of different fish species (Kaynar and Beyatli 2009) Additionally, research on PHA-producing mixed microbial cultures and recombinant strains across different reactor configurations highlights the potential for PHA production (De Schryver 2010a).
The production costs of Polyhydroxybutyrate (PHB) are significantly influenced by the yield obtained from the substrate, which depends on the bacterium's ability to accumulate PHB, the technological processes used, and the efficiency of recovery methods (Choi and Lee, 1999) Notably, up to 40% of the total costs in PHB production are attributed to the substrate, making it a key cost factor (Singh et al., 2013b) Additionally, Chanprateep (2010) provides pricing information for various commercial PHB products, highlighting the economic aspects of PHB production.
Table 1.3 PHB production (% on cell dry weight) by Bacillus species isolated from the intestines of various fish species in nutrient medium (NB – Merck) (Kaynar and Beyatli
Fish origins Bacillus species Yield of PHB (%)
Bacillus megaterium and Bacillus pasteurii are notable species within the Bacillus genus, which also includes Bacillus circulans, Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Bacillus coagulans, Bacillus sphaericus, Bacillus pumilus, Bacillus lentus, Bacillus badius, and Bacillus brevis These bacteria are recognized for their diverse applications in biotechnology and agriculture, highlighting their significance in various fields.
Bacillus badius, Bacillus licheniformis Bacillus licheniformis, Bacillus thuringiensis, Bacillus thuringiensis
Table 1.4 Accumulation of poly-β-hydroxybutyrate (PHB) in different types of bacterial cultures (after De chryver 2010a)
Strain/culture Carbon source PHB content
Production cost (€/kg PHB) Reference Pure strains (for commercial PHB production):
Glucose Glucose Fatty acids Lauric acid Sucrose
Choi and Lee 1997 Choi and Lee 1999 Choi and Lee 1999
Research by Choi and Lee (1999) and subsequent studies by Chen et al (2009) indicate that current production costs are likely to vary due to fluctuations in substrate costs and advancements in process optimization However, it is important to note that there is a lack of available data to support these findings.
PHB content (% on cell dry weight)
Production cost (€/kg PHB) Reference
Mixed cultures (emphasis on waste valorization):
Excess sugar Acetate Effluent of anaerobically fermented sludge Acetic, lactic and propionic acid Municipal wastewater Food waste
Castilho et al 2009 Johnson et al 2010 Cai et al 2009
Chua et al 2003 Rhu et al 2003
Cavalheiro et al 2012 Zhu et al 2010 a Current production costs can be expected to be different due to changes in substrate costs and process optimization; b no data available
Table 1.5 The current large volume manufacturers of polyhydroxyalkanoates (Chanprateep
Market price (in 2010) (€/kg) PHB
Nodax TM Nodax TM Kaneka PHBH Green Bio
Mitsubishi Gas Chemical Company Inc (Japan) Telles (USA)
Biomer Inc (Germany) Tianan Biologic, Ningbo (China)
P&P (USA) Lianyi Biotech (China) Kaneka Corporation (Japan) Tianjin Green Bio-Science Co/DSM (The Netherlands) Meredian (USA)
2.5 3.7 n/a n/a n/a n/a: price could not be found
To reduce the production cost of PHA, it is essential to enhance processing efficiency through optimization and large-scale production while utilizing cheaper substrates Current research focuses on various carbon sources, including starch-based materials, (hemi-)cellulolytic materials, and streams containing oils, fatty acids, or glycerol, as well as gaseous mixtures of H2 and CO2, and organic matter from waste and wastewater Notably, crude glycerol, a by-product of the biodiesel industry, has been explored for PHB production using different bacterial strains.
Paracoccus denitrificans and Cupriavidus necator JMP 134 (Mothes et al 2007), Burkholderia cepacia ATCC 17759 (Zhu et al 2010) and Bacillus firmus NII 0830 (Jincy et
35 al 2013) The mechanism of PHB production from crude glycerol is described in Figure 1.10
Figure 1.10 Biochemical pathway involved in bacterial production of PHB from glycerol
Thesis objectives and outline
In the past decade, poly-β-hydroxybutyrate (PHB), a natural compound derived from bacteria, has garnered significant interest among aquaculture researchers globally Despite its proven benefits for various aquaculture species, the high cost of PHB limits its widespread adoption in the industry Therefore, there is an urgent need to develop cost-effective production methods for PHB and explore its effective application in larviculture.
This thesis aimed to explore the application of amorphous PHB-accumulating bacteria in crustacean larviculture and to develop an innovative method for culturing these bacteria by integrating them with the production of Artemia nauplii.
Chapter 2 (Effect of Alcaligenes eutrophus carrier of poly- β -hydroxybutyrate for the culture of M rosenbergii larvae) :
The study examined the impact of feeding M rosenbergii larvae with a lyophilized form of A eutrophus, which contained two different levels of polyhydroxybutyrate (PHB) at 10% and 80% of cell dry weight (CDW) The objective was to assess how these PHB levels influenced the growth performance of prawn larvae and their resistance to disease when challenged with Vibrio harveyi BB120.
A eutrophus, cultivated in a standard nutrient medium with 12g/L salinity, effectively produces PHB and demonstrates significant protective capabilities for Artemia nauplii and M rosenbergii larvae against the pathogenic bacteria Vibrio campbellii.
LMG21363 and V harveyi BB120 was investigated by adding it into the culture water or by enriching the live food, respectively
Chapter 3 explores the alterations in glycerol, glycogen, trehalose, total organic carbon (TOC), and total nitrogen (TN) within the hatching medium of axenic decapsulated Artemia franciscana cysts during the incubation period.
40 measured In addition, it was attempted to use the carbon and nitrogen released into the hatching medium as a substrate for growing PHB accumulating bacteria
Chapter 4 focuses on the protection of gnotobiotic Artemia nauplii from the pathogenic Vibrio campbellii using Bacillus sp LT12, which was cultured in an axenic hatching medium of Artemia franciscana The study aimed to select Bacillus strains from shrimp and fish intestines based on their suitability as food for Artemia nauplii and their ability to produce polyhydroxybutyrate (PHB) Additionally, it investigated the accumulation of PHB in these Bacillus strains when cultured in the axenic medium and assessed whether this accumulation provides protection to Artemia nauplii against the pathogenic strain V campbellii LMG21363 when the bacilli are added to the culture medium.
Chapter 5 (PHB-accumulating Bacillus sp cultured in the axenic hatching medium of
This chapter examines the protective effects of Artemia franciscana on Macrobrachium larvae against Vibrio harveyi It specifically focuses on the impact of Bacillus strain LT12, which is cultured in axenic hatching medium of Artemia (AHMA) and enriched into Artemia nauplii.
Macrobrachium larvae challenged with Vibrio harveyi BB120 The Bacillus strain LT12 was grown in Artemia hatching medium in two different ways: the Bacillus was either
Artemia nauplii were either grown in axenic hatching medium and separated after 16, 20, or 24 hours of incubation, or directly co-cultured in the same medium throughout a 26-hour hatching process In the latter approach, the effect of adding supplemental glycerol at the beginning of incubation was also evaluated.
Figure 1.11 Setting of the different chapters in this research with PHB as a common denominator
Introduction
Bacterial infections significantly hinder the production of giant freshwater prawn (Macrobrachium rosenbergii), impacting both larviculture and post-larval stages Mass mortality in larvae and infections in post-larvae are primarily attributed to opportunistic pathogens, posing a serious challenge for prawn farming (Sung et al 2000; Kennedy et al 2006; Shailender et al 2012; Nhan et al 2010b).
Vibrio spp are the primary pathogens responsible for disease outbreaks in M rosenbergii larvae, highlighting the need for effective management in larviculture While maintaining optimal environmental conditions in prawn hatcheries is essential, the widespread use of antibiotics poses a risk of developing multiple resistances in pathogens, which can lead to ineffective treatments and potential resistance transfer to both animal and human pathogens To promote sustainable M rosenbergii culture, it is crucial to explore and develop new microbial control techniques.
Short chain fatty acids (SCFAs) are promising alternatives for controlling pathogenic bacteria due to their bacteriostatic and bactericidal properties, which depend on their concentration (Defoirdt et al 2009) These acids penetrate bacterial cell membranes in their un-dissociated form, leading to a release of protons (H+) in the cytoplasm and a decrease in intracellular pH As a result, bacteria must expend energy to eliminate excess protons, which hinders their growth and can even cause cell death (Kato et al 1992; De Schryver et al 2010b) In aquaculture, SCFAs have been shown to enhance the survival of Artemia nauplii during challenges with pathogenic Vibrio campbellii (Defoirdt et al 2006b), and other pathogens like luminescent vibrios and Salmonella spp are also affected by SCFAs (Defoirdt et al 2007b; Najdegerami et al.).
The use of short-chain fatty acids (SCFAs) in aquaculture presents challenges due to their high solubility in water, leading to low absorption efficiency by aquatic animals Consequently, this necessitates administering doses significantly higher than biologically required However, the recently introduced PHB strategy provides a promising solution to this issue, as PHB is synthesized to enhance the effectiveness of SCFAs in aquatic environments.
A diverse range of Gram-positive and Gram-negative bacteria utilize polyhydroxybutyrate (PHB) as an intracellular energy and carbon storage medium PHB is advantageous due to its water insolubility and its ability to be biologically degraded into β-hydroxybutyric acid.
Numerous studies have demonstrated the beneficial effects of PHB as a biocontrol compound in sustainable aquaculture The direct addition of crystalline PHB to the culture medium has shown protective effects against vibriosis in Artemia franciscana nauplii Furthermore, supplementing crystalline PHB in the diets of juvenile fish species, including European sea bass and Siberian sturgeon, has resulted in improved weight gain, survival rates, and specific growth rates Additionally, enriching Artemia nauplii or rotifers with crystalline PHB has been found to enhance the survival and growth performance of larval M rosenbergii and Chinese mitten crab.
A study by Nhan et al (2010b) demonstrated that the presence of vibrios in the gut of prawn larvae decreased when using PHB Despite the high cost associated with crystalline PHB particles derived from PHB-accumulated bacteria, amorphous PHB, which remains within the bacteria, has also been shown to provide beneficial effects.
The study aimed to evaluate the beneficial effects of amorphous PHB supplied through live food Artemia on M rosenbergii larvae, building on previous findings regarding the Artemia model system (Halet et al 2007; Cam et al 2009) and the cost-effectiveness of using bacteria rich in PHB as a biocontrol strategy in aquaculture Two tests were conducted: a non-challenged growth test and a challenge test using Vibrio harveyi Key assessment parameters included the survival and growth of the larvae, along with TCBS counts to estimate vibrios in the larval gut during the growth test, and survival rates and TCBS counts in the larval gut during the challenge test.
Materials and methods
2.1.2.1 Origin of Macrobrachium prawn larvae
Macrobrachium rosenbergii broodstock was imported from Vietnam and maintained in a freshwater recirculation system with a bio-filter To ensure optimal conditions, approximately 20% of the water in the broodstock tanks was replaced daily, following the removal of waste and uneaten feed Water quality parameters were carefully monitored, keeping NH4-N, NO2-N, and NO3-N levels below 0.2, 0.1, and 10.0 mg/L, respectively, in accordance with established guidelines The temperature was consistently maintained at 28 ± 1 °C using a heater, while the lighting regime provided an intensity of 8 μE/(m²·sec) with a 12-hour light and dark cycle The prawns were fed ad libitum twice daily with commercial pellet feed A single ovigerous female breeder was selected from the broodstock tank and isolated in a hatching tank for breeding.
The larvae were kept in a tank measuring 40 cm until they hatched, with management conditions identical to those of the broodstock tank Daily, fifty percent of the water was renewed, and the salinity was maintained at 6 g/L After hatching, the larvae that swam to the surface were collected for subsequent experiments (Cavalli et al 1999; Nhan et al 2010b).
2.1.2.2.1 Axenic hatching of Artemia franciscana
Experiments utilized Artemia franciscana as live food, with cysts sourced from the Great Salt Lake in Utah, USA (EG ® Type, INVE Aquaculture, Belgium) Bacteria-free cysts were prepared following the modified procedures of Marques et al (2004a), using filtered autoclaved artificial seawater containing 12 g/L of Instant Ocean synthetic sea salt The sterilized cysts were then resuspended in 1 L of this seawater, equipped with a 0.22 µm air filter for continuous sterile aeration The hatching process lasted 24 hours at 28 °C under constant illumination of approximately 27 μE/(m²·sec), resulting in the production of sterile nauplii.
2.1.2.2.2 PHB-accumulated Alcaligenes eutrophus and PHB particles preparation
Two batches of a PHB-accumulated A eutrophus strain (also known as Cupriavidus necator,
Ralstonia eutropha, and Wautersia eutropha), lyophilized and containing 10% PHB and
A eutrophus H16 was cultivated to produce 80% PHB on dry weight, designated as A10 and A80 The process began with a stock culture stored at -80 °C in cryovials containing glycerol and a late exponential-phase liquid culture in Luria-Bertani (LB) medium A 200 µL aliquot was inoculated into 5 mL of LB medium and incubated for 24 hours at 30 °C with shaking This culture was then subcultured into 100 mL of seeding medium, which included fructose and various nitrogen and phosphate sources, and incubated for another 24 hours The trace element solution used was filter sterilized and combined with autoclaved fructose and magnesium sulfate The resulting seed culture was then inoculated into 250 mL of cultivation medium, which contained similar ingredients, and incubated at 30 °C and 200 rpm to facilitate PHB production.
A 10 mL/L trace element solution was prepared similarly to the seed medium, with the pH adjusted to 6.80 using 5 M NaOH To prevent substrate limitation during cultivation, fructose was continuously added to the flasks, and its concentration was measured using the phenol-sulfuric acid method (Dubois et al 1956) Additionally, ammonium (NH4 +-N) levels were assessed colorimetrically with Hach Lange cuvette tests, as limited ammonium can shift carbon flux from biomass to PHB synthesis, leading to PHB accumulation Samples were collected at the end of the biomass growth phase (approximately 16 hours) and the PHB accumulation phase (approximately 30 hours) for analysis of dry cell mass and PHB concentration Cell concentration was determined by centrifuging 15-20 mL of culture broth at 7000 x g using a SORVALL RC6+ centrifuge (Thermo).
In a study conducted in Clintonpark Keppekouter, Belgium, cell pellets were prepared by centrifugation at 4 °C and subsequently washed and lyophilized to a constant weight The cell concentration was determined by measuring the weight difference between the tubes containing the pellets and empty tubes For PHB analysis, dried samples and external standards underwent methanolysis with 50% methanol and 50% NaOH, and the resulting 3-hydroxybutyric acid was analyzed via HPLC using 0.05% H3PO4 as the mobile phase The PHB content was calculated as a percentage of the PHB concentration relative to the total cell concentration, with A eutrophus cells accumulating 10% PHB at the end of the biomass growth phase (termed "A10") and reaching 80% PHB at the conclusion of the accumulation phase (termed "A80") Notably, the microbial cells were unviable after lyophilization, showing no growth upon inoculation into LB medium.
The crystalline PHB particles used in the trials (98% poly-β-hydroxybutyrate – 2% poly-β- hydroxyvalerate, Goodfellow, Huntingdon, England) were ground through a 30 àm sieve prior to use
2.1.2.2.3 Enrichment of axenic Artemia nauplii with PHB-accumulated A eutrophus and crystalline PHB particles
After 24 h of cyst incubation, axenically hatched Artemia nauplii (Instar II) were washed with 1 L FAASW The washed nauplii were enriched with PHB-containing A eutrophus in FAASW as indicated in Table 2.1.1 and 2.1.2 Crystalline PHB particles at a dose of 800 mg/L served as a reference treatment and resembled the amount of PHB in the treatment
In a study involving axenic Artemia nauplii, a concentration of 1000 mg/L A80 was used for enrichment, while a control group was treated similarly without PHB enrichment The density of the Artemia nauplii for enrichment ranged from 80,000 to 100,000 individuals per liter of filtered artificial seawater (FAASW) After a two-hour enrichment period, the nauplii were rinsed with clean freshwater before being provided ad libitum to M rosenbergii larvae.
Table 2.1.1: Overview of the treatments in experiment 1 – survival and growth test (each treatment was performed in quintuplet)
Treatment name Live food for
PHB concentration during enrichment (mg/L)
Control Non-enriched (axenic) Artemia nauplii / 0 0
PHB-800 mg/L Enriched Artemia nauplii Crystalline PHB particles 800 800
A10-100 mg/L Enriched Artemia nauplii A eutrophus (10% PHB) 100 10
A10-1000 mg/L Enriched Artemia nauplii A eutrophus (10% PHB) 1000 100
A80-10 mg/L Enriched Artemia nauplii A eutrophus (80% PHB) 10 8
A80-100 mg/L Enriched Artemia nauplii A eutrophus (80% PHB) 100 80
A80-1000 mg/L Enriched Artemia nauplii A eutrophus (80% PHB) 1000 800
Table 2.1.2 Overview of the treatments in experiment 2 – challenge test (each treatment was performed in quintuplet)
Treatment name Challenge Live food for
Control / Non-enriched (axenic) Artemia nauplii / 0
Non-enriched (axenic) Artemia nauplii Enriched Artemia nauplii
800 A10-100 mg/L + BB120 + V harveyi Enriched Artemia nauplii A eutrophus (10% PHB) 100
A10-1000 mg/L + BB120 + V harveyi Enriched Artemia nauplii A eutrophus (10% PHB) 1000
A80-10 mg/L + BB120 + V harveyi Enriched Artemia nauplii A eutrophus (80% PHB ) 10
A80-100 mg/L + BB120 + V harveyi Enriched Artemia nauplii A eutrophus (80% PHB) 100
A80-1000 mg/L + BB120 + V harveyi Enriched Artemia nauplii A eutrophus (80% PHB) 1000
The larval rearing experiments utilized large and small glass cones placed in a thermostatically controlled water bath, maintaining a temperature of 28 ± 1 °C Gentle aeration was provided to ensure a dissolved oxygen level exceeding 5 mg/L Additionally, a lamp system was installed above the cones to deliver 12-14 μE/(m²·sec) of light at the water surface for 12 hours each day.
For experiment 1 (survival and growth test), 35 large glass larval rearing cones containing
800 mL of 0.2 μm filtered brackish water (12 g/L salinity) were stocked with 60 M rosenbergii larvae of 1 day after hatching (DAH) The larvae were fed ad libitum twice a day
(9:00 and 17:00) for 20 days with enriched Artemia according to treatment (Table 2.1.1)
Every three days, we carefully siphoned out uneaten Artemia and waste from the cones, replacing 50% of the culturing water with freshly filtered brackish water to ensure the safety of the larvae.
In Experiment 2, a challenge test was conducted using 40 small glass cones, each containing 100 ml of FAASW, stocked with 25 M rosenbergii larvae of DAH 1 The larvae were fed ad libitum with enriched Artemia twice daily for 9 days, while being challenged with Vibrio harveyi BB120 on day 2 Prior to the experiment, Vibrio harveyi BB120 was preserved in 40% glycerol at -80 °C, and subsequently inoculated on an LB agar plate with 12 g/L salinity for 24 hours Two colonies from the agar plate were cultured in 25 mL of fresh LB medium (12 g/L salinity) for 24 hours at 28 °C with constant agitation at 120 rpm The optical cell density was measured spectrophotometrically at 550 nm, and the pathogenic bacteria were introduced into the M rosenbergii larval culture at a concentration of approximately 10^7 CFU per mL, with no water exchange occurring during the experiment.
2.1.2.4.1 Measurement of PHB content in enriched Artemia nauplii
Artemia nauplii enriched as live food for experiments 1 and 2 were carefully rinsed several times with de-ionized water and dried at 103 °C for 4 h A mortar was used to grind the dried
Artemia nauplii into powder Five mg of dried Artemia powder was incubated at 60 o C for 1 h with 0.5 mL sodium hypochlorite (5%) to break the cell walls of the dried Artemia nauplii
In a study by Aslim et al (1998), samples were centrifuged at 6,000 x g for 20 minutes and washed twice with a 1:1 acetone and ethanol mixture, followed by rinsing with deionized water To extract PHB, 5 mL of chloroform was added to the pellet, and the samples were incubated overnight at 28 °C on an orbital shaker at 250 rpm After evaporation of the extracts at 40 °C, 4 mL of concentrated sulfuric acid was added, and the mixture was heated at 100 °C for 20 minutes Once cooled, the absorbance at 235 nm was measured using a spectrophotometer, as described by Kaynar and Beyatli (2009), with PHB concentration determined from a standard curve of external PHB standards.
The percentage of PHB in the samples was calculated as:
PHB (%) = (PHB (mg) x 100 %) / CWD (mg)
2.1.2.4.2 Measurement of PHB content in enriched and purged Artemia nauplii
The study evaluated the impact of intestinal purging methods on the measurement of PHB in enriched Artemia nauplii Two purging techniques were employed: cellulose purging for 2 hours and starvation for 12 hours Following external washing with FAASW, the Artemia nauplii were subjected to a 2-hour exposure to 1.5 g/L cellulose in FAASW, as outlined by Niu et al.
2012), or to FAASW for 12 h After the purging period, the Artemia nauplii were washed with FAASW and the PHB content was measured as mentioned above
In experiments assessing M rosenbergii larval survival, evaluations were conducted on days 10 and 20 for the first experiment, and day 9 for the second To determine larval survival, the glass cone containing the larvae was gently poured into a white tray, where the larvae were manually collected and counted using a modified 1 mL pipette After counting, the larvae and water were returned to the cone, ensuring that the process was carried out with care to minimize stress on the larvae.
In experiment 1, larvae development was assessed by collecting sixty larvae from each treatment at days 10 and 20, with 12 larvae per replicate After measurement, the larvae were returned to their cones for further observation The determination of larval stages was based on morphological characteristics outlined by New (2002), and larval stage indexes were calculated following the methodologies of Mallasen and Valenti (2006) and Baruah et al (2009).
Where S i is larval stage (i = 1 – 12); n i is the number of larvae in stage S i ; and N is the total number of larvae observed
2.1.2.4.5 Bacteria in the gut of M rosenbergii larvae
Results
2.1.3.1 PHB content in Artemia nauplii
The PHB content in Artemia nauplii enriched for 2 hours with PHB-containing A eutrophus or PHB particles ranged from 9.8 ± 0.8% to 17.4 ± 1.3%, while non-enriched Artemia nauplii exhibited a PHB content of 8.9 ± 0.9%.
The study found that Artemia nauplii enriched with 800 mg/L PHB particles exhibited a significantly higher PHB content of 17.4 ± 1.3% compared to other treatments Additionally, the PHB content in the treatments A80-100 mg/L and A80-1000 mg/L was notably greater than that of the control group and the treatments A10-100 mg/L, A10-1000 mg/L, and A80-10 mg/L.
Table 2.1.3 PHB content in non-enriched axenic Artemia nauplii (control) and axenic
Artemia nauplii after 2 h enrichment with A eutrophus containing 10% PHB (A10), A eutrophus containing 80% PHB (A80) or PHB particles (PHB)
PHB in Artemia nauplii (% on dry weight)
2 h enrichment 2 h enrichment followed by 2 h cellulose purging
2 h enrichment followed by 12 h starvation Control 8.9 ± 0.9 a,1 9.4 ± 1.1 a,1 9.4 ± 0.2 a,1
Values prior to and after 2 h cellulose purging or 12 h starvation are given Values represent means ± SD of three Artemia nauplii sampled on three random days during the experiment
Values within the same column not sharing the same superscript letter are significantly different (P