Biofloc management with different flow rates for solids removal in the <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> BFT culture system

Different water flows for solids removal in the Litopenaeus vannamei BFT system were evaluated. One control (no solids removal) and two treatments using different water flows, high (3945 L h^?1—HF) and low (1750 L h^?1—LF), were used with no water replenishment after each process, and the total dry weight of the solids was measured. L. vannamei (0.18 ± 0.06 g; 350 individuals m^?2) were stocked in 35-m^?3 tanks. For 17 weeks, the physical and chemical parameters were maintained within the recommended. To keep the total suspended solids concentrations at approximately 500–600 mg L^?1, clarifying was performed. The average water volume flowed by clarifiers was significantly different (p < 0.05) between HF (205 ± 34 m³) and LF (114 ± 24 m³). There was a significant decrease (p < 0.05) in the final tank volume in HF (28.09 ± 0.92 m³) and LF (28.62 ± 1.38 m³) due to the clarifying. Before clarifying (initial sample) and at the end of experiment (final sample) were not significantly different (p > 0.05) for crude protein, moisture or ash. The crude lipid of the LF in the final period was significantly lower (p < 0.05) compared to others in both periods. The survival, productivity and food conversion ratio were significantly better (p < 0.05) in the HF and LF treatments compared to those of the control. The best shrimp performance was obtained with solids removal. The lower flow in the clarifier facilitated particle settling, allowing adjustment of the flow.     

Performance of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> postlarvae reared in indoor nursery tanks under biofloc conditions at different salinities and zero-water exchange

Shrimp farming at low salinity is a trend that will continue to grow globally. Performance of Litopenaeus vannamei postlarvae in the nursery at different salinities with a biofloc technology (BFT) system needs to be explored further, as the nursery is currently used as a transitional stage between the hatchery and grow-out ponds. Hence, this study evaluated the effect of seven salinity levels (2, 4, 8, 12, 16, 25, and 35 ‰) on the performance of L. vannamei postlarvae reared with a BFT system and zero-water exchange at 2000 org/m³. Additionally, this study evaluated the water quality of all salinity treatments. After 28 days of culture, the findings showed that, under biofloc conditions, salinity affected the performance of some variables of water quality in some cases, but only the combination of a high nitrite-N concentration (>4 mg/l) and low salinity (2 and 4 ‰) caused up to 100 % shrimp mortality in the first 2 weeks. In the rest of the treatments (8, 12, 16, 25, and 35  ‰), shrimp survival was >72 %. Shrimp mortality was affected by salinity, especially when it decreased from 35–25–16 to 12 and 8 ‰. The organisms reared at low salinities presented lower final weights and specific growth rate than those reared at higher salinities. An inverse relationship was shown between the ion concentration and the final weight of shrimp.     

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.     

The effects of cyclical temperature changes on growth and physiological status of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

The purpose of this study was to investigate the growth and physiological status of Litopenaeus vannamei subjected to one constant temperature (25°C) and four cyclical temperature change regimes (25 ± 1°C, 25 ± 2°C, 25 ± 3°C and 25 ± 4°C). The growth rates of shrimp at 25 ± 2°C or 25 ± 3°C were significantly higher than that at a constant temperature of 25°C. On the other hand, the growth rate in 25 ± 4°C regime was significantly lower than those in other regimes. The daily feed intake rate of shrimp at 25 ± 4°C was the lowest, and the food conversion efficiency was also significantly lower than those at 25 ± 2°C and 25 ± 3°C, respectively. The food conversion efficiency at 25 ± 2°C or 25 ± 3°C was significantly higher than those in other regimes. Thus, it can be inferred that the growth enhancement in the test shrimp at the suitable diel fluctuating temperatures was due to high food conversion efficiency. Studies of the physiological parameters showed that at 25 ± 4°C, the hemolymph glucose content of the test shrimp was the lowest, while the activity of PK in hepatopancreas was the highest, which indicated that the test shrimp at 25 ± 4°C was in a stressed condition. The hemolymph glucose content of the test shrimp at 25 ± 3°C was the highest, and the activity of HK in hepatopancreas was the lowest. These results indicated that the test shrimps at 25 ± 3°C were not in a stressed condition. Compared with the constant temperature regime, the expression of HSP70 in any of the four cyclical temperature change regimes was not significantly increased. The reason for this might be that the fluctuation amplitude of ± 4°C did not induce the increased expression of HSP70.     

Genetic diversity status of Pacific white shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>) using SSR markers in Iran

Genetic diversity is vital for the maintenance of genetic pool in cultured shrimps. In order to estimate the current status of genetic diversity in Litopenaeus vannamei shrimp in Iran, as an exotic species, a total of 45 individuals from Amiri and Gorgeaj farms in Jask port of Hormozgan province and one hatchery in Gomishan city of Golestan province, were detected using four microsatellite loci. The number of alleles per locus was 5–10, and the mean effective number of alleles (N _e) across populations and loci ranged from 4.834 to 5.148. The overall mean observed heterozygosity (H _o) ranged from 0.450 to 0.479, which was lower than the expected one (0.789–0.794). There was nothing remarkable about any of the allele frequencies across populations or loci. The mean inbreeding coefficient (F _IS) and pairwise genetic differentiation (F _ST) among populations were 41.6 % and 0.133, respectively. The three studied populations departed from Hardy–Weinberg equilibrium (HWE). Analysis of molecular variance revealed 14 % variability among and 86 % within populations. However, considering departing from HWE and the high F _IS and F, the moderate pairwise F _ST values, importance of introducing genetically diverse broodstock and monitoring to control inbreeding is discussed.     

The immune modulatory effect of oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> L.) essential oil on <Emphasis Type="Italic">Tilapia zillii</Emphasis> following intraperitoneal infection with <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

The current study aimed to evaluate the possible effect of Origanum essential oil on innate immune parameters as well as the hematological profiles of Tilapia zillii following challenge with Vibrio anguillarum. Fifty-four of Tilapia zillii weighing 180?±?10.2 g were randomly distributed into three identical closed recirculating seawater systems. The study included three groups (G1, G2, and G3) repeated in triplicates. Fish of the first two groups were fed on a basal diet without herbs, whereas fish of the last group were fed on a basal diet supplemented with Origanum essential oil at concentration 1 g kg^?1 for 15 days. Subsequently, fish of G2 and G3 subjected to a peritoneal inflammation by intraperitoneally injecting V. anguillarum (5.5?×?10⁵ CFU mL^?1), whereas fish of G1 injected with saline and served as control. Fish of all groups were then sampled at 4, 12, and 24 h post injection. No mortalities were observed in both basal and Origanum fed groups. However, some specimens of fish fed basal diet showed dorsal fin erosions, eroded mouth, and detached skin. Although the kinetics of RBCs, WBCs, Hb, and differential leukocyte values remained unchanged in fish fed different diets at the beginning of the trial, significant increases in those values were observed in fish fed Origanum essential oil at 12 and 24 h post injection. Similarly, an augmentation of plasma proteases, antiproteases, and lysozyme activities were recorded in fish fed Origanum essential oil at the same particular sampling points. A significant enhancement in plasma bactericidal capacity was only recorded in fish fed Origanum essential oil at 12 and 24 h post challenge compared to those fed basal diet. In conclusion, Origanum essential oil had a pronounced influence on the innate immunity and increased the fish resistance to V. anguillarum. These data gave insight into the potential use of Origanum in prophylactic strategies against threatening pathogens.     

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.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

Contribution of bioflocs to the culture of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> post-larvae determined using stable isotopes

The aim of this study was to use preselected quality indicators for Litopenaeus vannamei post-larvae and the stable isotopes technique with δ¹³C and δ¹⁵N to determine the influence of bioflocs in shrimp feeding during the nursery phase, between PL1 and PL30. A control treatment (CT) with water renewal was compared to a biofloc treatment (BT) that received organic carbon fertilizations. Different types of commercial feed (Stresspak and Flake-INVE?, PL40-GUABI?), microalgae (Chaetoceros muelleri) and Artemia sp. nauplii were used as food sources. The physical and chemical parameters of water and ammonia were monitored daily, and nitrite, nitrate and alkalinity were measured weekly. Suspended solids and the microorganisms of bioflocs were characterized. At the end of the experiment, fifteen shrimps of each replicate were collected to evaluate nine larvae quality indicators. Isotopic values of ¹³C and ¹⁵N of food sources and shrimp tissues were collected on days 10, 20 and 30, and a Bayesian model of isotopic mixture measured the contributions of these sources to the shrimp biomass. Salinity, alkalinity and nitrite differed significantly between the treatments but were appropriate for post-larvae production. The larvae quality conditions in the CT treatment were ranked as “good,” whereas the BT conditions were considered “excellent” according to the analysis of larval quality parameters. The stable isotopes analysis showed that the PL40 GUABI? feed was the most important food source throughout the experimental period in the CT. In the BT (10th and 20th days), the bioflocs and commercial feeds (Flake-INVE?, PL40-GUABI?) did not present significant variations in their contribution ratios; however, on the 30th day, the contribution of the PL40-GUABI? feed was higher when compared to bioflocs. In general, the commercial feed played a more important role as a food source for Litopenaeus vannamei larvae during the nursery phase, although the consumption of bioflocs led to a better larvae quality.     

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.     

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.     

The use of alternative prey (crayfish, <Emphasis Type="Italic">Procambarus clarki</Emphasis>, and hake, <Emphasis Type="Italic">Merlucius gayi</Emphasis>) to culture <Emphasis Type="Italic">Octopus vulgaris</Emphasis> (Cuvier 1797)

The effects of two alternative prey (crayfish and hake) were tested on growth and survival of both juveniles and adults of Octopus vulgaris in two experiments. Octopuses fed the control (squid) were larger (3.0 ± 0.7 kg) than those fed crayfish (2.4 ± 0.6 kg) at the end of experiment I. Similarly, overall growth rates were higher for octopuses fed squid (1.7 ± 0.3 and 1.2 ± 0.2 %BW day⁻¹, respectively). Average feeding rates for the experiment were not different, being 6.5 ± 0.9 and 7.5 ± 0.9 %BW day⁻¹, respectively, for octopuses fed either squid or crayfish. Nevertheless, food conversions for the experiment were higher (42.4 ± 2.7%) for octopuses fed squid compared to the ones fed crayfish (23.9 ± 1.9 g). For experiment II, hake and crayfish were compared to squid; the final weight of octopuses fed squid, hake or crayfish was 1,183.0 ± 242.7 g, 1,175.6 ± 240.1 g and 922.3 ± 160.1 g, respectively. Overall growth rates for the experiment were 1.9 ± 0.2 %BW day⁻¹, 1.9 ± 0.3 %BW day⁻¹ and 1.1 ± 0.3 g, respectively. Final weight and growth rates were never different (P > 0.05) between octopuses fed squid and hake, but were always higher (P < 0.05) compared to the ones fed crayfish. Average feeding rates for experiment II were similar for the three diets, and of 4.6 ± 1.5, 4.2 ± 1.3 %BW day⁻¹ and 5.1 ± 0.9 %BW day⁻¹, respectively, for octopuses fed squid, hake or crayfish. Food conversions for experiment II were of 41.0 ± 9.6%, 40.5 ± 9.9% and 21.3 ± 7.4 g, respectively, for octopuses fed squid, hake or crayfish, and were always higher for octopuses fed squid and hake compared to crayfish. The results indicate that crayfish is not an adequate replacement for the usual prey to fatten octopus, even considering its much lower market price.     

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.     

Anesthesia induces stress in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>), Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>) and Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

Stress in response to anesthesia with benzocaine, MS-222, metomidate and isoeugenol was studied in Atlantic salmon (Salmo salar), Atlantic halibut (Hippoglossus hippoglossus), and Atlantic cod (Gadus morhua) with no concomitant stress from handling or confinement in association with anesthesia or sampling. All of the anesthetics tested induced a stress response in all species, displayed by a release of cortisol to the water. MS-222 anesthesia elicited the highest cortisol release rates, reaching maximum levels 0.5 h post-exposure and returning to basal levels after 3–4 h. Benzocaine anesthesia caused a bimodal response where the initial peak in cortisol release rate was followed by a second increase lasting towards the end of the trial (6 h). This bimodality was more profound in Atlantic salmon than in Atlantic halibut and Atlantic cod. Metomidate anesthesia induced the lowest release of cortisol of the agents tested in both Atlantic halibut and Atlantic cod, but resulted in a bimodal response in Atlantic salmon where the initial increase in cortisol release was followed by a larger increase peaking at 2–2.5 h post exposure before returning to basal levels after 5 h. The stress induced in Atlantic salmon by isoeugenol anesthesia resembled that of MS-222, but did not reach the same elevated level. Overall, the cortisol release was most profound in Atlantic salmon followed by Atlantic halibut and Atlantic cod.     

Demographic and competition studies on <Emphasis Type="Italic">Brachionus ibericus</Emphasis> and <Emphasis Type="Italic">Proales similis</Emphasis> in relation to salinity and algal (<Emphasis Type="Italic">Nannochloropsis oculata</Emphasis>) density

We isolated the rotifers Brachionus ibericus and Proales similis from the sediment of shrimp tanks and studied their individual demographic characters and competition between them at two food levels (0.25?×?10⁶, 1.00?×?10⁶ cells ml^?1 of Nannochloropsis oculata at 25 °C) and salinities ranging from 10 to 30‰. Our hypothesis was that growth rates would be higher with increasing food levels and salinities. Observations were taken twice a day for life table studies and daily once for population growth experiments. Using survivorship and fecundity data, we derived various life history variables. Although the average life span (7.6?±?0.4 days) and gross reproductive rate (33.8?±?2.9 neonate female^?1 day^?1) of B. ibericus were higher than those of P. similis (average life span 5.4?±?0.6 days and gross reproductive rate 13.0?±?0.6 neonate female^?1 day^?1), the population growth experiments showed that P. similis had higher r values (0.32?±?0.005 day^?1) than B. ibericus (0.23?±?0.002 day^?1) at 1.0?×?10⁶ cells ml^?1 of N. oculata. The rotifer P. similis was more adversely affected due to the presence of B. ibericus than vice versa. The data are important for developing techniques for a large-scale culture of these rotifers as food in aquaculture.     

Digestive enzymes and in-vitro digestibility of different species of phytoplankton for culture of the freshwater pearl mussel, <Emphasis Type="Italic">Hyriopsis</Emphasis> (<Emphasis Type="Italic">Hyriopsis</Emphasis>) <Emphasis Type="Italic">bialatus</Emphasis>

Hyriopsis (Hyriopsis) bialatus has been cultured during the mussel life cycle from glochidia to the adult stage with a low total survival of 6% up to 130-day-old juveniles. The main digestive enzymes (amylase and proteinases) were not detectable in one-day-old juveniles, and increased during development. The stomach, including digestive glands, was the major digestive organ for both carbohydrate and protein. The optimum conditions for amylase activity were 40°C and pH 7; for acidic proteinases they were 60°C and pH 5. Two main alkaline proteinases were found in the intestine, with optimum conditions of 30°C and pH 8 and 60°C and pH 8. To improve mussel survival by finding suitable phytoplankton species and age as food for juveniles and adults, an in-vitro digestibility test was performed on ten algal species three and seven days old using amylase and proteinases in crude enzyme extracts from different mussel life stages. Among the phytoplankton selected, the three most efficiently digested by juveniles were seven-day-old Chlorella sp.2, seven-day-old Chlorococcum sp. and seven-day-old Kirchneriella incurvata, in the ratio 1:1:3 for 30-day-old juveniles and 3:1:1 for 130-day-old juveniles. For the adult mussel, three-day-old Chlorella sp.2, seven-day-old Coccomyxa sp., and seven-day-old Monoraphidium sp., in the ratio 3:1:1, were the most digestible phytoplankton. Levels of in-vitro digestibility were related to the quality (not the concentrations) of carbohydrate and protein in the phytoplankton mixtures, and protein digestibility seemed to be the key factor determining food quality for the mussel.     

Gene structure and expression analyses of multiple vitellogenesis-inhibiting hormones in the whiteleg shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

In the whiteleg shrimp Litopenaeus vannamei, the existence of six sinus gland peptides (SGPs) possessing vitellogenesis-inhibiting hormone (VIH) function has been previously demonstrated. Genomic and cDNA sequence information for two of these is already known. Here, we cloned cDNAs for the remaining three, i.e., those encoding Liv-SGP-A, -B, and -F. The deduced amino acid sequences were comprised of a signal peptide, a CHH precursor-related peptide, a mature peptide, and a C-terminal amidation signal. The genomic DNA for Liv-SGP-A, -B, -C, -F, and -G, was found to be organized as three exons and two introns. The insertion of the two introns were conserved in all VIH genes, with intron I being inserted six residues following the signal peptide, and intron II after the 40th residue of the mature peptide. We also quantified the relative simultaneous expression of Liv-SGP-A, -B, -C, -F, and -G in relation to molting and unilateral eyestalk ablation. The expression levels of each gene in the eyestalks of adults did not change significantly during molting or following unilateral eyestalk ablation. However, unilateral eyestalk ablation significantly decreased expression levels at 10 and 20 days for Liv-SGP-A and -C, and at 20 days for Liv-SGP-G in the eyestalks of subadults. This study has therefore clarified the genomic structure of the five major subtype I VIHs in L. vannamei, and elucidated their expression levels in the eyestalks in relation to molting and unilateral eyestalk ablation.