Enhancement of <Emphasis Type="Italic">Penaeus monodon</Emphasis> shrimp postlarvae growth and survival without water exchange using marine <Emphasis Type="Italic">Bacillus pumilus</Emphasis> and periphytic microalgae

We have investigated the possibility of using a consortium of marine bacterium and periphytic microalgae to improve the water quality and increase the growth and survival of the shrimp Penaeus monodon in a hatchery system. Three treatments were evaluated for their effect on P. monodon postlarvae (PL) when the culture water was not changed: Bacillus pumilus alone (B); periphytic microalgae alone (M); B. pumilus + periphytic microalgae (BM). P. monodon PL raised in a tank of unchanged water without bacterium and periphytic microalgae served as the control. The water in tanks of the M and BM treatments had significantly low levels of total ammonia-nitrogen (TAN) (0.03 and 0.01 mg l⁻¹, respectively) and nitrite-nitrogen (NO₂-N) (0.03, 0.01 mg l⁻¹, respectively) than that in the B (TAN 0.80, NO₂-N 0.68 mg l⁻¹) and control (TAN 1.11, NO₂-N 1.12 mg l⁻¹) tanks. Moreover, PL cultured in tanks M and BM had significantly higher survival and specific growth rates and a significantly higher resistance to the reverse salinity stress test than those in the B and control tanks. Compared to the control PL, the PL cultured in the BM tanks had significantly higher levels of protein, lipid, polyunsaturated fatty acids, ecosapentaenoic acid, and docosahexaenoic acid. The culture water in tanks BM also contained significantly less Vibrio than the control water. Our results illustrate the beneficial effects of a B. pumilus and periphytic microalgae consortium on improving the water quality and the growth and survival of shrimp PL grown in a hatchery system.     

Assessment of growth condition for a candidate probiotic, <Emphasis Type="Italic">Shewanella algae</Emphasis>, isolated from digestive system of a healthy juvenile <Emphasis Type="Italic">Penaeus monodon</Emphasis>

To conquer disease problem in shrimp industries, probiotic biocontrol is a well-known remedy now. The antagonistic ability of separated isolates from different parts of juvenile P. monodon was screened against shrimp Vibrio pathogens, V. parahaemolyticus and V. alginolyticus. The most antagonistic effect was observed for an isolate that primarily identified as Shewanella algae using conventional method followed by Biolog GN and GP microplates. Since adaptability to the host optimum cultural condition of the target organism is of the great importance, response surface methodology, with central composite design, was applied to assess log cell count response of S. algae in different incubation conditions. Therefore, four independent variables were assumed as: temperature (10–50°C), pH (6–10), NaCl concentration (0–50‰) and time (12–60 h). The coefficients of multiple determinations (R ²) for the responses log cell count of S. algae being 0.827. Temperature was the merely significant independent variable that affected the log cell count of the candidate probiotic. The candidate probiotic was revealed a reasonable growth response in quite wide range of temperature, pH and NaCl concentration in which the maximum levels were in same range of optimum shrimp culture.     

Detection of prostaglandin E<Subscript>2</Subscript> in polychaete <Emphasis Type="Italic">Perinereis</Emphasis> sp. and its effect on <Emphasis Type="Italic">Penaeus monodon</Emphasis> oocyte development in vitro

Prostaglandins are involved in the reproductive processes in a variety of animals, including crustaceans. It was found that polychaetes, the best maturation diet for shrimp broodstock, possessed the greatest variation of prostaglandin E₂ (PGE₂) when compared with other live feeds. The level of PGE₂ varied according to sizes, feed intake, sources and type of polychaete. The matured and also larger sand polychaete Perinereis sp. contained higher PGE₂ levels than younger and smaller sand polychaetes (18.16 ± 5.82 ng PGE₂ mg⁻¹ protein for polychaetes at an average length of 10 cm up to 160.8 ± 37.09 ng PGE₂ mg⁻¹ protein for polychaetes at an average length of 17 cm). The PGE₂ levels in ovaries and haemolymph of female shrimp fluctuated with the developmental stage of the ovaries. The highest concentration of PGE₂ in haemolymph was at stage 3 of ovarian development, whereas the highest concentration of PGE₂ in shrimp ovaries was at stage 4. In vitro incubation of Penaeus monodon pre-vitellogenic oocytes with polychaete extract and synthetic PGE₂ demonstrated that both PGE₂s enhanced oocyte development, especially during late development and ovulation. The putative role of PGE₂ from polychaetes or the presence of PGE₂ in polychaetes may be a factor in their role as a dietary constituent required for shrimp oocyte development.     

Isolation and characterization of Noctiluca-killing bacteria from a shrimp aquaculture pond in Thailand

To control harmful algal blooms (HABs), in particular dinoflagellate Noctiluca scintillans, which causes damage to shrimp production, algicidal bacteria were isolated from shrimp pond water and screened for their killing effect against N. scintillans under laboratory condition. Among 260 bacterial isolates, 10 showed killing effects on N. scintillans. Out of 5 strains showing relatively stronger killing activity, 4 strains belonged to the Gammaproteobacteria group. Among these, strain BS2 (identified as Marinobacteria salsuginis) showed the strongest Noctiluca-killing activity. In the present study, we also investigated bacterial effects against growth of other phytoplankton species. Strain BS2 killed only N. scintillans but not any other phytoplankton (Heterosigma akashiwo NIES-6, Chatonella antique NIES-1, Chaetoceros ceratosporum, Prorocentrum lima, and Dunaliella sp.). Results of the present study lead us to expect prevention of damage by HABs, in particular dinoflagellate N. scintillans, in shrimp farming and mitigation for shrimp production in ponds using Noctiluca-killing bacteria.     

Antibacterial effect of medium-chain fatty acid: caprylic acid on gnotobiotic <Emphasis Type="Italic">Artemia franciscana</Emphasis> nauplii against shrimp pathogens <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">V. parahaemolyticus</Emphasis>

The present study was carried out to determine the antibacterial effect of caprylic acid in the culture system of Artemia franciscana nauplii inoculated with shrimp pathogens Vibrio harveyi and V. parahaemolyticus. To begin with, the antibacterial effect of different concentrations (1, 10 and 100 mM) of caprylic acid against V. harveyi and V. parahaemolyticus was assessed through bacterial growth study. This indicated that at 100 mM concentration, the growth of the pathogens was completely inhibited within 5 h, whereas, in 1.0 and 10 mM concentrations, effective inhibition was observed with the extension of time. Subsequently, the influence of pH variation on the growth inhibitory effect of 10 mM caprylic acid against V. harveyi and V. parahaemolyticus at different pH (5–7) was also made through bacterial growth study. At pH 5, the pathogen growth was very less, compared with pH 6 and 7. The Artemia nauplii (instar II) reared in 10 mM caprylic acid supplemented medium were challenged with V. harveyi and V. parahaemolyticus, and the mortality was recorded at an interval of 6 h up to 60 h. In this study, the mortality of Artemia nauplii reduced to 20.61 and 16.30% in V. parahaemolyticus and V. harveyi challenged groups, respectively. The present results provide evidence for the potential antibacterial activity of caprylic acid in aquaculture against luminescent vibrios.     

Enhancement of survival and metamorphosis rates of <Emphasis Type="Italic">Penaeus monodon</Emphasis> larvae by feeding with the diatom <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis>

The purpose of this study was to compare the performance of two species of diatoms, Thalassiosira weissflogii and Chaetoceros gracilis, in the larviculture of the black tiger shrimp Penaeus monodon. Shrimp larvae were fed with either C. gracilis, T. weissflogii, or a combination of the two species of diatoms. The larvae fed solely with T. weissflogii or a combination of the two types of diatom had significantly higher survival rates and faster metamorphosis than those fed solely with C. gracilis. The numbers of diatom cells consumed by larvae during 3-h periods were determined, revealing that larvae consumed significantly higher numbers of C. gracilis than T. weissflogii. However, when the protein, total fatty acid, and polyunsaturated fatty acid content of the two species of diatom are compared, significantly higher amounts of each are found in T. weissflogii. Converting the number of diatom cells consumed into equivalent protein, total fatty acid, eicosopentaenoic acid, and decosahexaenoic acid reveals that larvae fed with T. weissflogii received significantly higher amounts of all the nutrients, compared to those consuming C. gracilis. The results showed an advantage of feeding T. weissflogii to C. gracilis in enhancing survival and metamorphosis in P. monodon larvae.     

Isolation and characterization of <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> causing infection in farmed <Emphasis Type="Italic">Portunus trituberculatus</Emphasis> in China

In August 2008, a massive epizootic occurred among Portunus trituberculatus reared together with Palaemon carincauda Holthuis and Sinonovacula constricta in the seawater pond of Ningang farm, Rudong district, Jiangsu province, China. The disease occurred in crabs from juveniles to adults, and the mortality rate reached 30–40%. The diseased crabs exhibited lethargy, hepatopancreas turgidity, and elevated levels of turbid hemolymph. A gram-negative, rod-shaped bacterium (designated as strain LGS-1) was isolated from the ascitic fluid of the diseased and moribund crabs and was confirmed as the causative agent by our infectivity study. We conducted morphological and biochemical characterization of LGS-1, and sequenced the 16S rRNA gene, which led us to identify the bacterium as Vibrio metschnikovii. Drug sensitivity tests showed that this pathogenic bacterium is sensitive to florfenicol, orfloxacin, and SXT, but completely resistant to antibacterial drugs like gentamicin, erythrocin, and acheomycin. This is the first report on Vibrio metschnikovii as a virulent pathogen for Portunus trituberculatus.     

Recent expansion of Northeast Atlantic and Mediterranean populations of <Emphasis Type="Italic">Melicertus</Emphasis> (<Emphasis Type="Italic">Penaeus</Emphasis>) <Emphasis Type="Italic">kerathurus</Emphasis> (Crustacea: Decapoda)

We analysed the genetic diversity of Melicertus kerathurus (Penaeidae), a commercially valuable penaeid shrimp that is distributed in the Mediterranean Sea and eastern Atlantic Ocean. We examined the polymorphism of a 494 bp DNA segment of the mitochondrial COI region in 173 individuals, sampled in nine Mediterranean and two Atlantic samples, covering the whole range of the species from the tropical waters of the Gulf of Guinea to the eastern part of the Mediterranean Sea. The mean nucleotide and haplotype diversities were π = 0.00275 and h = 0.718, respectively, for the global data set, with the highest values occurring in the African samples and the lowest in the Adriatic Sea. A clear sample differentiation was found (F _st = 0.194), but this did not reflect a geographical pattern and there were only faint traces of an Atlantic–Mediterranean subdivision. Mismatch analysis and a high significant negative value of Tajima’s D suggested that M. kerathurus is not at mutation drift-equilibrium, but underwent a recent expansion after a period of low effective sample size. A postglacial recolonisation of the Mediterranean from an Atlantic refuge could be hypothesised based on these data.     

Potential of cinnamon (<Emphasis Type="Italic">Cinnamomum verum</Emphasis>) oil to control <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In this study, four essential oils—cinnamon oil, leech lime oil, lemongrass oil, and turmeric oil—were examined for their antimicrobial activities against Streptococcus iniae, a bacterium that is pathogenic in fish, in which it causes streptococcosis. Cinnamon oil was the most potent antimicrobial agent among these oils, with a minimal inhibitory concentration (MIC) of 40 μg/ml. By using gas chromatography–mass spectrometry (GC–MS), it was found that the major components of cinnamon oil were cinnamaldehyde (90.24), limonene (2.42%), cinnamyl acetate (2.03%), linalool (1.16%), and α-terpineol (0.87%). Of these compounds, only cinnamaldehyde exhibited antimicrobial activity against S. iniae, with an MIC of 20 μg/ml. In an in vivo trial, no mortality was apparent in fish fed on fish diets supplemented with 0.4% (w/w) of cinnamon oil and with 0.1% (w/w) of oxytetracycline 5 days prior to infection with S. iniae. These results indicate that cinnamon oil had a protective effect on experimental S. iniae infection in tilapia, and thus has the potential to replace the antibiotics used to control this disease.     

Growth and survival of grouper <Emphasis Type="Italic">Epinephelus</Emphasis><Emphasis Type="Italic">coioides</Emphasis> (Hamilton) larvae fed free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> at first feeding

The free-living nematode, Panagrellus redivivus, was tested as live food for grouper Epinephelus coioides larvae during the first feeding stage. A series of experiments were conducted to determine the acceptability of the free-living nematodes in grouper larvae at first feeding, the optimum nematode density and the response of the larvae to nutritionally enriched nematode. All experiments were conducted in 200-L conical tanks filled with 150-L filtered seawater and stocked at 15 larvae L⁻¹. Duration of feeding experiments was up to day 21 (experiment 1) and 14 days (experiment 2 and 3). Brachionus plicatilis and Artemia (experiment 1) and Brachionus plicatilis alone (experiment 2 & 3) was used as the control treatment. Observations indicated that the grouper larvae readily fed on free-living nematodes as early as 3 days posthatching, the start of exogenous feeding. Optimum feeding density for the larvae was 75 nematodes ml⁻¹. The enrichment of cod liver oil or sunflower oil influenced the total lipids and n-3 highly unsaturated fatty acids of P. redivivus, which in turn influenced those of the grouper larvae, however, growth and survival of the larvae were not affected (P > 0.05). The results from this investigation showed that the nematode, P. redivivus, can be used as first live food for grouper larvae from the onset of exogenous feeding until they could feed on Artemia nauplii.     

Effect of <Emphasis Type="Italic">Cratoxylum formosum</Emphasis> on innate immune response and disease resistance against <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> in tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The effect of aqueous extract of Cratoxylum formosum on innate immune response and disease resistance in tilapia Oreochromis niloticus was investigated. The fish were fed diets containing 0% (control), 0.5% (diet 1), 1% (diet 2), and 1.5% (diet 3) of C. formosum aqueous extract for 30 days. At the end of the experimental period, parameters of innate immune response including phagocytosis of blood leukocytes, lysozyme activity in plasma, and respiratory bust activity were examined. Feeding the fish with diet 2 and diet 3 for 20 days enhanced phagocytic activity and for 30 days stimulated lysozyme and respiratory burst activities. Diet 1 increased the phagocytic activity at 30 days, but did not affect the other measured parameters. All parameters were not significantly changed (P > 0.05) in the control group throughout the experiment. Following 30 days of feeding, fish were infected with S. agalactiae. The cumulative mortalities of bacterial-infected tilapia that were fed diet 1, diet 2, and diet 3 were 56, 12, and 10%, respectively, compared with 85% in the control group. These results indicate that the aqueous extract of C. formosum may elevate the innate immune response and enhance disease resistance in tilapia.     

Effect of VP28 DNA vaccine on white spot syndrome virus in <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry. Considering the global environmental, the economic and sociological importance of shrimp farming, and the constraints of high intensity cultivation, development of novel control measures against the outbreak of WSSV become inevitable. In this study, we have explored the protective efficacy of DNA vaccination and tissue distribution of the recombinant plasmid in immunized Litopenaeus vannamei. The VP28 gene was cloned in the eukaryotic expression vector pVAX1, and the construct vector was named as lpv28. The protective effect of lpv28 against WSSV was evaluated in L. vannamei by injecting lpv28 construct and later challenging with WSSV. Expression of these proteins from the recombinant plasmids was confirmed in vitro by RT-PCR and Western blot analysis. The result of vaccination trials showed that a survival rate in shrimp vaccinated with lpv28 was 52.5% at most compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups showed that the vaccinated groups owned a high level of lysozyme, alkaline phosphatase, and total superoxide dismutase when compared to the control group. Furthermore, protein expression analysis indicated that VP28 can be detected in gill, muscle and head soft tissue of the shrimps in the immunized group after 14th day injection. Thus, the result indicated that DNA vaccination strategy has a potential utility against WSSV.     

Use of the freshwater rotifer <Emphasis Type="Italic">Brachionus angularis</Emphasis> as the first food for larvae of the Siamese fighting fish <Emphasis Type="Italic">Betta splendens</Emphasis>

Larvae of the Siamese fighting fish Betta splendens were reared on the mass-cultured small freshwater rotifer Brachionus angularis Laos strain (UTAC-Lao), Paramecia sp., and Artemia as live food sources. Larvae fed live food were found to have a significantly high survival rate (97.5–100%) 18 days after hatch (DAH) in comparison to the control unfed larvae, which died by 12 DAH. Rotifer-fed larvae were found to grow faster than paramecia-fed larvae. The fastest growth rate was observed in larvae fed a combination of rotifer and Artemia, with growth in these larvae increasing by 282% by 18 DAH [total length (TL) 11.3 ± 1.2 mm] relative to body measurements taken 3 DAH. The next fastest growth rate was observed in rotifer-fed larvae, with a 158% increase in growth observed by 18 DAH (TL 7.6 ± 0.5 mm). The paramecia-fed larvae were found to grow by only 54.3% (TL 4.6 ± 0.1 mm) during the same period.     

<Emphasis Type="Italic">Neoheterobothrium hirame</Emphasis> (Monogenea) alters the feeding behavior of juvenile olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We monitored feeding behavior and survival of starved juvenile olive flounder experimentally infected with the gill monogenean Neoheterobothrium hirame. Infected flounder increased amount of the time spent in the water column by 117% when trying to capture live mysids, Neomysis sp. They also showed different feeding patterns from those of uninfected fish and made fewer attacks towards prey during one feeding attempt. Although the average numbers of mysids captured by individuals were similar between infected and uninfected fish, heavily infected fish tended to catch less prey. These results indicate that N. hirame reduces the feeding efficiency of the host for capturing live prey and possibly makes them more vulnerable to predation during feeding. We could not detect any obvious difference in survival rates between uninfected, lightly and heavily infected fish during 3 months of starvation. There was no evidence that starvation makes fish more susceptible to N. hirame. The present study provides first experimental evidence that N. hirame affects feeding behavior of juvenile olive flounder and supports the idea that this parasite indirectly reducing the host’s survival and may be responsible for the recent reduction of the flounder population in Japan.     

Optimisation of larval culture of the mussel <Emphasis Type="Italic">Mytilus</Emphasis><Emphasis Type="Italic">edulis</Emphasis> (L.)

The blue mussel Mytilus edulis is a commercially important species whose fishery and culture generally relies on natural spat collection. Hatchery-production could provide an alternative source of seed, enabling reliable expansion of the industry. Mussel spawning and larval rearing trials were carried out to optimise elements of hatchery production. Culturing fertilised eggs at low density (20–200 eggs cm⁻²) rather than high density (400–720 eggs cm⁻²) significantly improved the quality of first veliger larvae and differences in this improvement were evident between the eggs from different females (maternal effects). Veliger larval growth at 17 or 21°C was significantly faster than growth at 14°C. Feeding veliger larvae an identical total ration either daily or at 2–3 day intervals did not significantly affect their growth. Different microalgal diets (1: Isochrysis sp. (clone T-ISO), 2: Chaetoceros calcitrans forma pumilus, 3: C. muelleri, 4: mixed Isochrysis sp. (clone T-ISO) and C. calcitrans f. pumilus, and 5: mixed Isochrysis sp. (clone T-ISO) and C. muelleri) were tested on veliger larval growth and mixed diets outperformed single-species diets.     

Effect of microencapsulated thyme essential oil on white spot virus-infected <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

The present work was performed to investigate if feeding Litopenaeus vannamei with microencapsulated thyme essential oil (TEM, 1.05 g thyme essential oil per 100-g powder) adsorbed on commercial pellet feed was able to protect shrimps against white spot syndrome virus (WSSV) disease. Five treatments were tested: uninfected shrimp fed with commercial pelleted feed (T_C, negative control), WSSV-infected shrimp fed with commercial pelleted feed (T₁, positive control), WSSV-infected shrimp fed with commercial pelleted feed with 0.1% TEM (T₂), WSSV-infected shrimp fed with commercial pelleted feed with 0.5% TEM (T₃), and WSSV-infected shrimp fed with commercial pelleted feed with 1% TEM (T₄). At 72 h post infection, phenoloxidase activity of shrimps treated with 1% TEM did not show significant differences with T_C values but it was significantly higher than that of the other treatments (T₁, T₂, and T₃). Moreover, shrimps treated with T₄ presented absence of clinical signs of WSSV infection and their survival rate was significantly higher than that of T₁, T₂, and T₃ treatments. Therefore, 1% TEM seemed to protect shrimps against WSSV symptoms. Using microencapsulated thyme essential oil may help to fight against WSSV in shrimp farms.     

Experimental nursery culture of the mud crab <Emphasis Type="Italic">Scylla paramamosain</Emphasis> (Estampador) in China

For the improvement of nursery culture of the mud crab Scylla paramamosain, a special nursery facility was purposely designed and built. This facility consists of nursery ponds (areas of 20, 50, and 120 m², respectively) and a seed collecting pond connected to each nursery pond. There were 140 nursery facility units in operation for large-scale juvenile crab production. A series of experiments were carried out using the facility to investigate the effects of the age of megalopae, stocking density, and culture duration of both megalopae and first-stage crabs on the survival of megalopae and early juveniles of S. paramamosain. The results showed that when megalopae were stocked at a density between 3,000 and 5,000 ind m⁻² and reared to reach the first-stage crabs, a survival rate of up to 50% could be achieved. When the first-stage juvenile crabs were stocked at a density between 2,000 and 3,000 ind m⁻², their survival was more than 50% after 14 days of culture.     

Induction of diploid gynogenesis in turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> with left-eyed flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig’s effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm⁻². At 15°C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3°C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1°C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.     

Effects of culture density and bottom area on growth and survival of the cuttlefish <Emphasis Type="Italic">Sepia</Emphasis><Emphasis Type="Italic">officinalis</Emphasis> (Linnaeus, 1758)

The effects of culture density and bottom areas on cuttlefish (Sepia officinalis) culture were studied. Cuttle fish were cultured under three experimental combinations of culture density and bottom area: (1) high density and small bottom surface area; (2) low density and large bottom area; (3) high density and large bottom area. Each experimental protocol was repeated in triplicate. Average weights at the end of the experiment were of 65.8 ± 5.8, 87.1 ± 5.6 and 78.7 ± 5.9 g for cuttlefish cultured under the conditions of protocol 1, 2, and 3, respectively; these differences were significant between each of the three groups. Total biomass increased faster (up to 7.5 kg per tank) under the high density/large bottom area conditions (protocol 3) due to the larger number of animals and low mortality in those tanks. Growth rates (%bw day⁻¹) were different between protocols, with growth rates of 2.1 ± 0.1, 3.0 ± 0.2, and 2.5 ± 0.1%bw day⁻¹ obtained for cuttlefish cultured according to protocol 1, 2, or 3, respectively. Average feeding rates were similar for the three groups—10.7 ± 0.8, 9.7 ± 2.0, and 9.6 ± 1.1%bw day⁻¹ for cuttlefish cultured according to protocols 1, 2, and 3, respectively, while food conversions (%) were different—21.5 ± 3.2, 32.4 ± 2.5, and 27.0 ± 1.1%bw day⁻¹, respectively. Total mortality was high in the high density/small bottom area tanks, 30%, while it was very low for the groups cultured under conditions of low and high density/large bottom area, 4%. Based on these results, we conclude that culture conditions that provide large bottom areas also provide good survival conditions and promote growth in comparison those with small bottom areas, even under conditions of lower culture densities.