Growth,absorption and assimilation efficiency by mature cuttlefish (<Emphasis Type="Italic">Sepia officinalis</Emphasis>) fed with alternative and artificial diets

The effects of feeding three natural frozen diets, grass shrimp (Palaemonetes sp.), crayfish (Procambarus clarkii) and fish (Sardina pilchardus) and two semi-humid artificial diets (based on fish powder) to mature cuttlefish, Sepia officinalis, were analysed. Growth and feeding rates (GR and FR, % BW day⁻¹), food conversions (FC, %), and total protein and lipid composition of the diets were determined. Digestive gland to body weight ratio and absorption efficiency were calculated for each diet. Cuttlefish fed shrimp and crayfish grew larger (1.5 and 1.1% BW day⁻¹, respectively) compared to the other diets. Shrimp promoted the highest FC, followed by crayfish, and sardine. The highest FR was obtained for cuttlefish fed crayfish (8.4% BW day⁻¹). Although both artificial diets were accepted, none produced growth. A positive correlation (r = 0.96) between cuttlefish ingestion rate and digestive gland weight was obtained. Some cannibalism occurred among cuttlefish fed the artificial diets during the last week of the experiment. According to the results obtained, P. clarkii could be used as an alternative prey to shrimp for rearing adult mature (>50 g) S. officinalis.     

Effects of Using Live Grass Shrimp (Palaemonetes varians) as the only Source of Food for the Culture of Cuttlefish, Sepia officinalis (Linnaeus, 1758)

The life cycle of cuttlefish fed ad libitum exclusively on live grass shrimp (Palaemonetes varians) was studied during 5 consecutive generations. Different culture temperatures promoted different (P < 0.05) exponential growth for each life cycle, being summer generations shorter than those of winter. Higher temperatures promoted higher IGR’s and mortality, while lower temperatures promoted increased life span, reproduction stages, total fecundity and total egg biomass. Increased generations also seemed to increase fertility. A “hybrid” generation promoted the best results in terms of hatchling weight, individual fecundity and fertility. Mean egg weight was related to female size and embryonic development took longer at lower temperatures. Brood stock sex ratios seemed to be temperature related. All of these culture aspects were also compared between themselves in order to establish future brood stock methodologies. Grass shrimp proved to be a good diet for the culture of cuttlefish throughout the life cycle. The use of only one species reduces costs and labor associated to cuttlefish culture.     

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.     

<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.     

Repeatable immersion infection with <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis> reproducing clinical signs and moderate mortality

Pseudotuberculosis is a bacterial septicaemia caused by Photobacterium damselae subsp. piscicida in several marine fish species. Yellowtail Seriola quinqueradiata is the most sensitive fish species to this disease. The internal organs of naturally infected yellowtail exhibit whitish spots, tubercle-like tissue structures, consisting of bacterial accumulations. There have been many trials for experimental infection, however adequate method of infection that reproduces moderate mortality and primary clinical signs has not yet established. Present investigation evaluated an immersion infection method by using logarithmic culture-phase bacteria resulting in higher mortality than that using stationary culture-phase bacteria. Typical white spots on the spleen and kidney were also observed constantly in dead fish. Transmission electron microscopy and fluorescent antibody microscopy showed bacterial clusters not only in the spleen and kidney but also in the blood channels in the secondary gill filaments. These results were confirmed repeatedly by plural experiments. The use of logarithmic-phase bacteria in immersion infection is an appropriate technique to reproduce moderate mortality and primary clinical signs, which will be a reliable infection method also for the challenge test of pseudotuberculosis vaccine.     

Effect of a probiotic bacterium <Emphasis Type="Italic">Bacillus circulans</Emphasis> PB7 in the formulated diets: on growth,nutritional quality and immunity of <Emphasis Type="Italic">Catla catla</Emphasis> (Ham.)

Bacillus circulans PB7, isolated from the intestine of Catla catla, was evaluated for use as a probiotic supplement in the feeds for the fingerlings of Catla catla. The effect of supplement on the growth performance, feed utilization efficiency, and immune response was evaluated. Catla fingerlings (ave. wt. 6.48 ± 0.43 g) were fed diets supplemented with 2 × 10⁴ (feed C1), 2 × 10⁵ (feed C2), and 2 × 10⁶ (feed C3) B. circulans PB 7 cells per 100 g feed for 60 days at 5% of the body weight per day in two equal instalments in triplicate treatments. The control feed (CC) was not supplemented with the B. circulans. All the feeds were isocaloric and isonitrogenous. Fish fed with feed C2 displayed better growth, significantly (P ≤ 0.05) highest RNA/DNA ratio, a lower feed conversion ratio (FCR), and a higher protein efficiency ratio (PER) than the other experimental diets. Highest carcass protein and lipid was also observed in the fish fed C2 feed compared to the others. Significantly (P ≤ 0.05), highest protease was recorded in fish fed feed C2 (47.9 ± 0.016) and lowest in fish fed feed C3 (32.10 ± 0.009), where α-amylase activity did not differ significantly (P ≤ 0.05) beyond the lowest inclusion level. ALP, ACP, GOT, and GPT in the liver of Catla catla were the highest (P ≤ 0.05) in fish fed C2 feed. The highest TSP, albumin, and globulin was observed in fish treated with C2 feed after 60 days feeding trial, but the lowest glucose level was observed in the same treatment. After the feeding trial, the non-specific immunity levels and disease resistance of fish were also studied. Phagocytic ratio, phagocytic index, and leucocrit value were the highest in fish fed feed C2. After the feeding trial, the fish were challenged for 10 days by bath exposure to Aeromonas hydrophila (AH1) (10⁵c.f.u. ml⁻¹ for 1 h, and, after 7 days, 10⁷c.f.u. ml⁻¹ for 1 h). Highest survival percentage was observed in fish fed with feed C2 compared with only 6.66% in the controls, which indicated the effectiveness of B. circulans PB 7 in reducing disease caused by A. hydrophila.     

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 soy sauce <Emphasis Type="Italic">koji</Emphasis> and lactic acid bacteria on the fermentation of fish sauce from freshwater silver carp <Emphasis Type="Italic">Hypophthalmichthys molitrix</Emphasis>

The effects of soy sauce koji and the lactic acid bacterium, Tetragenococcus halophilus, were studied on the fermentation of fish sauce prepared from Chinese silver carp. The fish sauce prepared without koji and the lactic acid bacterium contained low levels of organic materials, total nitrogen, and organic acids. The use of koji was effective in increasing these qualitative parameters and further improved the amino acid score of the fish sauce. Addition of T. halophilus had an effect on lowering the pH value during the initial period of fermentation when the soy sauce koji was also supplemented. In contrast, T. halophilus-like bacteria were found to be predominant for all tanks fermented under the different starting conditions. Although it was not examined whether the T. halophilus-like bacteria observed after fermentation were the same as the starter-bacterium or not, it was suggested that T. halophilus plays an important role in the successful fermentation of silver carp fish sauce. Sensory evaluation conducted with Japanese and Chinese panelists also suggested the superiority of the use of koji for fermentation of silver carp fish sauce.     

Response of a Juvenile Cyprinid,Lake Minnow <Emphasis Type="Italic">Eupallasella perenurus</Emphasis> (Pallas), to Different Diets

Three commercial starters (Carp Starter, Uni Starter and Perla Plus) and one non-commercial, with frozen Chironomidae larvae as a reference diet, were evaluated for the intensive rearing of juvenile lake minnow Eupallasella perenurus, a cyprinid fish that is critically endangered in Poland. The growth, condition, survival, body deformities, and chemical body composition were studied. The 90-day laboratory experiment was performed at 22 °C with fish that were initially 24.6 mm (mean total lenth (TL)) and 0.11 g (mean body weight (BW)). Satisfactory fish growth was attained with all of the diets; however, the largest (p ≤ 0.05) final size (48.5 mm TL, 1.55 g BW) and the lowest condition coefficient (K = 1.34) were noted in fish fed the non-commercial starter. The final survival rates were very high (97.5–100%). Skeletal deformities (in 74 to 92% fish) were recorded exclusively in fish fed commercial starters. All commercial starters resulted in considerably higher lipid content and lower ash content than did the non-commercial starter and the reference diet. This suggests that both these factors might be responsible for body deformities. The present results proved that only the non-commercial starter is suitable for juvenile E. perenurus rearing under controlled condition, and that none of the commercial starters can be recommended.     

Circadian melatonin release <Emphasis Type="Italic">in vitro</Emphasis> by European sea bass pineal

Melatonin production by the fish pineal is under the control of photoperiod. In addition, in some fish species pineal melatonin production is self-sustained by an intrapineal circadian pacemaker, not in salmonids, though. The aim of this research was to test melatonin release in vitro by the pineal of sea bass held under LD 12:12 and DD conditions to determine the endogenous nature of melatonin rhythm. Circadian rhythmicity ( = 28.3 h) was found since rhythmic melatonin release persisted on DD.     

Age and growth of two gizzard shads, <Emphasis Type="Italic">Nematalosa come</Emphasis> and <Emphasis Type="Italic">N. japonica</Emphasis>, in coastal waters around Okinawa Island,southwestern Japan

The age and growth of two Nematalosa species around Okinawa Island were examined using sectioned otoliths collected from September 2003 to April 2006. Monthly changes in the frequency of the appearance of a translucent band on the outer margin of the otoliths indicated that ring formation occurred once a year from January to July for Nematalosa come and from January to March for Nematalosa japonica. The von Bertalanffy growth equations for both species were as follows: N. come: L _t  = 365.5{1 − exp[−0.111 × (t + 0.288)]} for females and L _t  = 214.7{1 − exp[−0.700 × (t – 1.110)]} for males; N. japonica: L _t  = 205.1{1 − exp[−1.068 × (t − 1.180)]} for females and L _t  = 195.5 {1 − exp[−1.293 × (t − 1.269)]} for males. The maximum ages observed for N. come and N. japonica were 11 and 6 years old, respectively. The growth of these species was characterized by the slow growth of N. come over many years, resulting in a larger size than N. japonica.     

Effect of Dietary Lipids on Macrophage Function,Stress Susceptibility and Disease Esistance in Atlantic Salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

As the supply of marine fish oil is becoming a limiting factor in the production of Atlantic salmon (Salmo salar), new diets and alternative sources of energy are being tested. Plant oils are natural potential candidates to replace fish oil, but the different levels of essential polyunsaturated fatty acids may influence the health and growth of salmon. In this study, we have investigated the resistance to transport stress and bacterial infection, phagocytic activity in head kidney macrophages and eicosanoid metabolism in salmon fed three different diets. In high-energy fishmeal based diets, 50% and 100% of the supplementary fish oil (FO) was replaced with soybean oil (SO). The three dietary groups were fed for 950 day-degrees at 5 °C (27 weeks) and 12 °C (11 weeks) before challenging the fish with Aeromonas salmonicida, analyzing the lipid composition of head kidney and examining macrophage function in vivo and in vitro. Dietary fatty acids affected the lipid composition of the kidney. The level of eicosanoid precursor’s 20:4n-6 and 20:3n-6 were 3 and 7-fold higher in the 100% SO group compared with the FO group. The total fraction of n-3 lipids in kidney was 19% in the SO group, compared to 16% and 12% in the 50% or 100% SO groups, respectively. However, the production of leucotriene B₄ (LTB) and prostaglandin E₂ (PGE) immunoreactive materiel from exogenously added arachidonic acid in head kidney macrophages was only affected by the composite diet (increased) at 5 °C. In addition, the phagocytic activity of kidney macrophages in vivo and in vitro was not affected by diet. No effect of diet was observed on transport stress or susceptibility to a bacterial infection with Aeromonas salmonicida. Atlantic salmon therefore seems to tolerate a diet solely based on soybean oil as lipid source, without any detrimental effects on growth, health and immune functions.     

Intraspecific diversity of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Harvey) Suringar (Laminariales,Phaeophyta) from Japan,China and Korea,based on the <Emphasis Type="Italic">cox1</Emphasis> gene and ITS2 sequences

As a trial to develop a method of authenticating the place of origin of circulated Undaria pinnatifida products, we investigated their intraspecific genetic diversity using the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) and the internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal DNA (rDNA) sequence. Four dried U. pinnatifida products labeled with their origins (one from Japan, one from China and two from Korea), natural plants collected from three locations (two from Japan and one from China), and cultivated plants collected from two locations (one from Japan and one from China) were used in the present study. The amplified fragments of cox1 were 664 bp in length, and the aligned sequences were highly homologous. Among the nine sequences, no insertions or deletions were found and six substitution positions were detected, and they were classified into five haplotypes. In contrast, multiple highly variable regions were found in ITS2, and some of them carried a restriction site for Mbo II. Polymerase chain reaction-restriction fragment length polymorphism analysis showed different restricted profiles among the tested samples. The availability of molecular markers for authenticating food products of U. pinnatifida is discussed.     

Assessment of reference dietary amino acid pattern for juvenile red sea bream, <Emphasis Type="Italic">Pagrus major</Emphasis>

Four semi-purified diets, containing crystalline amino acids (CAAs), were fed to juvenile red sea bream, Pagrus major in order to ascertain the ideal dietary amino acid pattern for this species. A control diet containing 50% casein–gelatin as protein sources, but no CAAs were fed to the fish. The other diets contained 30% casein–gelatin and 20% CAAs. CAAs were added to diets to simulate with amino acid pattern of the red sea bream eggs protein (REP), red sea bream larvae whole body protein (RLP), red sea bream juvenile whole body protein (RJP), and brown fishmeal protein (BFP). The juveniles (average initial body weight, 1.58 ± 0.01 g) were maintained in triplicate tanks and fed twice daily for 30 days. The highest weight gain was observed in juveniles fed the RJP diet. No significant difference was observed in juveniles fed the RLP and BFP diet. Feed efficiency ratio, protein efficiency ratio and amino acid retention in the whole body were significantly (p < 0.05) affected by the simulated dietary amino acid patterns. The essential amino acid profile and A/E ratios of the whole body after the growth trial showed little difference among the dietary treatments. The results suggest that red sea bream juveniles are able to utilize high amounts of CAA in coated form. The amino acid pattern of RJP could be used as an appropriate of reference dietary amino acid for this species.     

Circadian locomotor activity in Japanese sea catfish <Emphasis Type="Italic">Plotosus lineatus</Emphasis>

To find out whether or not the locomotor activity of the Japanese sea catfish, Plotosus lineatus, shows a circadian rhythmicity, the locomotor activity of this catfish was recorded in a laboratory. All of the catfish recorded (n = 20) showed a nocturnal locomotor activity rhythm under a light–dark (LD) cycle (LD 12:12). The locomotor activity started after the light was turned off, and declined during the period of darkness. In five of the six catfish we examined, locomotor activity shifted gradually over the course of 3–4 days in order to synchronize to the LD cycle after it was advanced by 6 h. Locomotor activity persisted within a constant darkness (DD) in all eight of the catfish. The average free-running period of locomotor activity under DD lasted for 24.2 ± 0.4 h (mean ± SD). Thus, the present results demonstrate that the Japanese sea catfish has an endogenous circadian oscillator entrained by an LD cycle.     

The effects of temperature in the life cycle of two consecutive generations of the cuttlefish Sepia officinalis (Linnaeus, 1758), cultured in the Algarve (South Portugal)

We are presently culturing the 4^th generation of thecuttlefish, Sepia officinalis in our laboratory. A firstgeneration (F1) was grown from eggs collected from the wild (Ria Formosa–South Portugal) during the summer, at mean temperatures of 27°C ± 3°. In the present study, a second generation(F2), originated from eggs laid in the laboratory by females from F1 wascultured between the start of autumn and the end of spring, at meantemperaturesof 15 °C ± 4 °C. The life cycle ofcuttlefish from F2 was compared to F1. Populations of 30 cuttlefish were usedineach experiment. Cuttlefish were grown from one day old until the cycle wascompleted (when the last female in each population had died). Cuttlefish fromF2cultured at much lower temperatures had a longer life cycle, of almost 9 months(260 days) compared to cuttlefish from F1, which completed their cycle in lessthan 6 months (165 days). Cuttlefish from F2 grew significantly larger (U =0.00; p < 0.01) with mean weights of 343.3 ± 80.5 g and248 ± 33.1 g for males and females, respectively, comparedtoF1 (199.6 ± 40 g and 143.3 ± 30.9 g formales and females, respectively). Females from F2 had higher fecundity (225eggsfemale^–1) compared to females from F1 (144 eggs perfemale^–1), produced bigger eggs (t = 45.60752; p < 0.0001),weighing 0.74 ± 0.18 g, compared to 0.46 ± 0.11 fromF1,and bigger hatchlings (t = 7,144783; p < 0.0001), weighing 0.10 ±0.02g, compared to 0.09 ± 0.02 g for the summerpopulation.