Reproductive biology of the commercial sea cucumber <Emphasis Type="Italic">Holothuria spinifera</Emphasis> (Echinodermata: Holothuroidea) from Tuticorin,Tamil Nadu,India

The annual reproductive cycle of the commercial sea cucumber Holothuria spinifera was studied in Tuticorin, Tamil Nadu, India, from September 2000 to October 2001, by macroscopic and microscopic examination of gonad tubule, gonad index and histology of gametogenic stages, to determine the spawning pattern. The gonad consists of long tubules with uniform development. It does not confirm the progressive tubule recruitment model described for other holothurians. The maximum percentage of mature animals, gonad and fecundity indices, tubule length and diameter, with the observations on gonad histology, ascertained that H. spinifera had the peak gametogenic activity during September and October 2001 followed by a prolonged spawning period from November 2000–March 2001.     

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.     

Immune response of sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> to <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> antigens

ABSTRACT:   Seawater fishes are affected by a pathology commonly called 'myxobacteriosis', caused by Tenacibaculum maritimum (formerly Flexibacter maritimus ). The disease is characterized by fin erosion and necrotic ulcers of skin and muscle, and by low but constant mortality in cultured marine fish; in Italy is one of the most important and widely spread diseases affecting sea bass Dicentrarchus labrax , gilthead seabream Sparus aurata , sharp-snouted bream Diplodus puntazzo , white bream Diplodus sargus , and six-tooted bream Dentex dentex . In order to obtain an effective vaccine against the disease, formalin killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) preparations were obtained from the T. maritimum strain SPVId and injected intraperitoneally twice into the sea bass. The fish immune response to the preparations was studied: agglutinating antibody titer and in vitro phagocytosis were determined after the first and second injection in order to evaluate whether the preparations are immunogenic or not and if the booster effect took place. The results show that FKC and LPS preparations increased the antibody titer after the first injection when compared to the control sea bass. Moreover, all the preparations stimulated a secondary (booster) response. In vitro phagocytosis of the total blood was significantly higher for all the preparations when compared to the controls, but the crude LPS immunized sea bass showed the highest activity.     

Spawning induced by cubifrin in the Japanese common sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

The neuropeptide cubifrin-I and its derivative cubifrin-L have recently been shown to be potent substances that induce in vitro oocyte maturation and spawning in the Japanese common sea cucumber Apostichopus japonicus. We have examined the reproductive behavior of A. japonicus in vivo following the injection of cubifrin-L into the body cavity with the aim of determining the practical value of using this peptide to induce spawning in the hatchery setting. The responsiveness of ovarian fragments with oocytes >155 μm in diameter to cubifrin-L in vitro was well correlated with the spawning success induced in vivo by a cubifrin-L injection. Mature sea cucumbers injected with cubifrin-L displayed sequential reproductive behaviors, which comprised climbing up the side wall of the tank toward the water surface, waving of the head, and shedding of gametes. Gamete shedding started about 60 and 80 min after the injection of cubifrin-L in males and females, respectively, and was completed almost simultaneously in the two sexes about 2 h post-injection. Repeated injections of cubifrin-L at intervals of about 10 days successfully induced multiple spawnings in males and females. These results demonstrate that cubifrin-L can be used as an effective inducer of spawning in Japanese sea cucumber cultivation.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

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.     

Immunological effects of glucan and <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> GG,a probiotic bacterium,on Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> intestine with oral <Emphasis Type="Italic">Aeromonas</Emphasis> challenges

The present study histologically examined the effects of glucan-containing and Lactobacillus rhamnosus GG (LGG)-containing diets on intestinal damages inflicted on Nile tilapia by Aeromonas challenges. Tilapia were fed control, glucan, and LGG diets for 2 weeks and were subsequently challenged with Aeromonas. The intestines were then histologically examined at 1, 7, 14, and 21 days post-infection. Mortality following the challenge was lower for the fish fed the glucan and LGG diets. The intestines of these groups also showed increased inflammatory cell infiltration and reduced intestinal damage from Aeromonas. Moreover, inflammatory cell infiltration occurred more rapidly in the glucan-fed than in the LGG-fed fish following the challenge. Before the challenge, the dominant mucous cell was the acid type in all the tests. After the challenge, the main mucus cell type in the proximal intestine of the glucan-fed fish shifted to AB-PAS double-staining cells, while in the LGG-fed fish, it remained the acid type throughout the test period, and the number of double-staining cells was smaller than in the control fish after the challenge. Thus, the different mucous cell and inflammatory cell responses show that glucan and LGG might have different immunostimulative effects, although they both reduced the intestinal damage following Aeromonas challenges.     

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.     

Reproductive cycle of yellow croaker <Emphasis Type="Italic">Larimichthys polyactis</Emphasis> in southern waters off Korea

The reproductive cycle and batch fecundity of yellow croaker Larimichthys polyactis were examined in 981 specimens (286 males, 695 females) caught from waters around Mokpo, Busan, and Jeju-do (Korea) between February 2008 and January 2009. Males and females with ripe-stage gonads were collected between April and June, at which time the mean gonadosomatic indices were also high, indicating that the spawning season of yellow croaker around the waters off southern Korea occurs from April to June. Degenerating postovulatory follicles as well as oocytes in the migratory-nucleus stage or hydrated stage were found in ripe-stage gonads, suggesting that this species can spawn repeatedly in a season. Batch fecundity in 45 females with migratory-nucleus or hydrated oocytes ranged from 19,396 to 106,311 eggs in fish varying from 20.0 to 26.7 cm in total length and was found to be positively related to total length and body weight. Our findings should assist in implementing a proper stock assessment of yellow croaker around the waters off Korea.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

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.     

Homogeneity of <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> from farmed amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> in Japan

Streptococcus dysgalactiae strains have been isolated from cultured amberjack Seriola dumerili and yellowtail Seriola quinqueradiata in Japan. To characterize the fish isolates, we performed genetic analysis and compared the biochemical properties of these isolates with those of the S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis strains isolated from mammals. The genetic analysis revealed that the fish isolates were genetically very similar to each other with high DNA–DNA relatedness (>95.4%) and sequence homology. Meanwhile, the DNA relatedness between mammalian isolates and the fish isolates was 73.4–82.6%. In biased sinusoidal gel electrophoresis (BSFGE) analysis, the restriction patterns of mammalian isolates were different from those of fish isolates. The fish isolates did not show streptokinase activity in plasminogen obtained from mammals. These characteristics enabled us to distinguish between the fish isolates and the Sdd and Sde strains isolated from mammals. In order to obtain epidemiological information on the fish isolates, BSFGE patterns from 284 S. dysgalactiae strains from fish in Japan were examined. Based on the results of BSFGE analysis, the fish isolates were classified into 16 groups (AP1–AP16) with restriction enzyme ApaI. The dendrogram based on BSFGE analysis indicated that all fish isolates using in this study were closely related.     

Reproductive cycle of yellow sea bream <Emphasis Type="Italic">Dentex tumifrons</Emphasis> in Wakasa Bay,the Sea of Japan off central Honshu

ABSTRACT:   To determine the spawning season and reproductive cycle of the yellow sea bream, Dentex tumifrons , in Wakasa Bay, in the Sea of Japan off central Honshu, seasonal change in the gonadosomatic index ( GSI ) was examined and histological observations of both ovaries and testes were conducted. The female GSI gradually increased from May, maintained a high level between August and October and then rapidly decreased. The histological observations of the ovary showed that vitellogenesis began from May and developed until September. Spent fish began to be observed in October and all the fish collected in December were spent. There was only one peak in the seasonal change in GSI of males and females in Wakasa Bay. Thus, yellow sea bream spawn only in autumn in Wakasa Bay. Differences in the reproductive cycle and number of spawning seasons were found between the two different latitudinal areas, Wakasa Bay and the East China Sea (low latitudinal area) where the spawning seasons were spring and autumn. An increase in water temperature is an important factor to initiate gonadal maturation (recrudescence) and is terminated by the short day-length in Wakasa Bay.     

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.     

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.     

<Emphasis Type="Italic">Virgibacillus proomii</Emphasis> and <Emphasis Type="Italic">Bacillus mojavensis</Emphasis> as probiotics in sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) larvae: effects on growth performance and digestive enzyme activities

This study examined the effects of two probiotics (Virgibacillus proomii and Bacillus mojavensis) on the digestive enzyme activity, survival and growth of Dicentrarchus labrax at various ontogenetic stages in three separate experiments. These probiotics were incorporated as single or mixed into fish feed for a period of 60 days. The growth parameters, proximate composition of whole body, digestive enzymes and gut microbiology were monitored at regular. The increments in length and weight and the survival were significantly higher (P < 0.05), and the values of food conversions were significantly lower (P < 0.05) in fishes fed the probiotic. The administration of V. proomii and B. mojavensis in diet resulted in an increase (P > 0.05) in body ash and protein content and in the specific activity of phosphatase alkaline and amylase in the digestive tract of all the fishes. V. proomii and B. mojavensis persisted in the fish intestine and in the feed in high numbers during the feeding period (group 1: 5.8 × 10⁴ CFU/ml, group 2: 9.6 × 10⁴ CFU/ml, and group 3: 9.8 × 10⁴ CFU/ml day 60). The two probiotics V. proomii and B. mojavensis were adequate for improved growth performance and survival and for healthy gut microenvironment of the host.