High yield and rapid growth of Neoparamoeba pemaquidensis in co-culture with a rainbow trout gill-derived cell line RTgill-W1

Neoparamoeba pemaquidensis is an ubiquitous amphizoic marine protozoan and has been implicated as the causative agent for several diseases in marine organisms, most notably amoebic gill disease (AGD) in Atlantic salmon. Despite several reports on the pathology of AGD, relatively little is known about the protozoan and its relationship to host cells. In this study, an in vitro approach using monolayers of a rainbow trout gill cell line (RTgill-W1, ATCC CRL-2523) was used to rapidly grow large numbers of N. pemaquidensis (ATCC 50172) and investigate cell-pathogen interactions. Established cell lines derived from other tissues of rainbow trout and other fish species were also evaluated for amoeba growth support. The amoebae showed preference and highest yield when grown with RTgill-W1 over nine other tested fish cell lines. Amoeba yields could reach as high as 5 x 10(5) cells mL(-1) within 3 days of growth on the gill cell monolayers. The amoebae caused visible focal lesions in RTgill-W1 monolayers within 24 h of exposure and rapidly proliferated and spread with cytopathic effects destroying the neighbouring pavement-like cells within 48-72 h after initial exposure in media above 700 mOsm kg(-1). Disruption of the integrity of the gill cell monolayers could be noted within 30 min of exposure to the amoeba suspensions by changes in transepithelial resistance (TER) compared with control cell monolayers maintained in the exposure media. This was significantly different by 2 h (P < 0.05) compared with control cells and remained significantly different (P < 0.01) for the remaining 72 h that the TER was monitored. The RTgill-W1 cell line is thus a convenient model for growing N. pemaquidensis and for studying host-pathogen interactions in AGD.     

Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel (Anguilla japonica) for its use in aquaculture

In the present study, a potential Lactobacilli probiotics were isolated from Japanese eels (Anguilla japonica) and characterized and evaluated for their possible use in eel farming. Sixteen Lactobacilli were isolated from intestines of Japanese eels, using selective media. The lactobacilli strains (represented as PL1 to PL16) were screened by their ability to produce digestive enzyme. Among these, three strains (PL11, PL13 and PL16) producing four digestive enzymes (amylase, cellulase, protease and phytase) simultaneously were characterized further using API ZYM kit. From these, PL11 (Lactobacillu (L.) pentosus) was identified as potential probiotics candidate producing 15 enzymes among 20 tested. Further examination of biological activities of PL11 revealed tolerance against pH, artificial bile juice and antibacterial activity against several fish pathogenic bacteria. The in vitro competitive exclusion assay also revealed 88.4% reduction in adhesion of fish pathogen (Edwardsiella tarda) by PL11 to host intestinal mucus. In vitro incubation of Japanese eel foregut with Baclight‐labelled PL11 showed colonization of the enterocyte surface by confocal and scanning electron microscopy. In summary, PL11 isolated from eels could serve as a potential probiotics with acid and bile tolerance, production of digestive enzymes, antibacterial activity and inhibition of fish pathogen adhesion to intestinal mucus.     

Disturbance‐induced responses of VHF and satellite tagged harbour seals

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Observations on growth, sexual maturity and spawning performance of pond-reared Penaeus merguiensis

This 12‐month preliminary study investigated the development of sexual characters, primary sexual maturity, ovarian maturity and spawning performance of pond‐reared Penaeus merguiensis in relation to culture conditions in south‐east Queensland, Australia. Post‐larvae of P. merguiensis were produced and cultured in two 60‐m³ tanks during the first 14 weeks. Before winter, they were harvested and stocked in three different overwintering facilities: a 200‐m² covered pond, two 60‐m³ outdoor tanks and a 15‐m³ indoor, recirculated tank at a stocking density of 10 individuals m^?2. The development of sexual characters was found to be similar to that reported previously in wild P. merguiensis. Males matured at younger ages and smaller sizes than females. Overall, the average size at primary sexual maturity of pond‐reared P. merguiensis was 23.1 mm carapace length for males (possessed spermatophores) and 29.3 mm for females (being impregnated). Water temperature and the availability of natural food strongly influenced prawn growth, maturity rate and their subsequent spawning performance. Growth, maturity rate and spawning performance of prawns in the covered pond were significantly higher than in the other overwintering facilities. Prawns started mating at 6–7 months, reached full ovarian maturation and spawned as early as about 8 (peaked at 9–11) months from hatching, producing high fecundity and viable larvae. There was a strong relationship (P < 0.001) between prawn size and fecundity. The results of this study suggest a potential for using pond‐reared broodstock P. merguiensis for hatchery production and for domestication or selective breeding programmes.     

Effect of Artificial Diets on Growth,Survival and Condition of Adult Cuttlefish, <Emphasis Type="Italic">Sepia officinalis</Emphasis> Linnaeus, 1758

The effects of artificial diets on growth and body condition of adult cuttlefish, Sepia officinalis were tested in two experiments. Supplemented prepared diets (fish myofibrillar protein concentrate) were fed during a 30-day and a 21-day experiments. Growth, feeding rate and food conversion of group-reared cuttlefish were analyzed. The first of these experiments tested four artificial diets, made with increasing levels of lysine, on adult cuttlefish. According to the chemical analysis, diets 1–3 had limiting concentrations of lysine and other essential amino acids (compared to mantle composition of the cuttlefish), while diet 4 was the only one where almost all essential amino acids were present in concentrations similar or higher than the ones present in cuttlefish mantle. A second experiment was conducted by isolating 16 adult cuttlefish individually, and feeding them the same four artificial diets, in order to obtain individual data. During Experiment 1, only the diet with the best chemical score (diet 4) produced growth (p < 0.05), with a mean instantaneous growth rate (MIGR) of 0.30% wet body weight (BW) d⁻¹. Similarly, individually reared cuttlefish fed diet 4 produced the highest IGR’s (0.26, 0.38 and 0.48% BW d⁻¹) and grew larger (p < 0.01). Comparison of cuttlefish fed the artificial diets vs. thawed shrimp and unfed cuttlefish indicated that cuttlefish fed the artificial diets were in an intermediate state. Growth rates obtained with the artificial diets (<0.4% BW d⁻¹) were considerably lower compared to natural prey, live or frozen, reported by other authors.     

Natural bixin as a potential carotenoid for enhancing pigmentation and colour in goldfish (Carassius auratus)

Discovering natural carotenoids for colour enhancement and health benefits of fish is important to develop new feed formulations. We have purified natural bixin from achiote seeds and evaluated the effect of colour enhancing and pigmentation in goldfish. Varying levels of bixin‐based diets were prepared with 420 g kg^?1 of crude protein and 120 g kg^?1 of lipid content. Our results clearly showed that bixin (0.05, 0.10, 0.20 and 0.60 g kg^?1) based diets significantly (P < 0.05) enhanced the skin and fin colour at 30 and 60 days compared to diet without bixin. Interestingly, diet which contains 0.20 g kg^?1 bixin and commercial feed (with astaxanthin) had similar effect on carotenoid deposition in skin. Moreover, total carotenoid deposition in fin was higher than in skin of all bixin‐containing diets. However, 0.60 g kg^?1 bixin‐containing diet had lower specific growth rate (1.01 ± 0.01) and higher feed conversion ratio (2.05 ± 0.19) compared to the control group. The present results demonstrate that achiote bixin can be successfully used as an alternative natural carotenoid source against synthetic astaxanthin in fish feed. Our data indicate that 0.20 g kg^?1 is a suitable dietary level of bixin to ensure strong pigmentation, acceptable growth and feed utilization in goldfish.     

Distribution and migration of Japanese common squid, Todarodes pacificus, in the southwestern part of the East (Japan) Sea

Data on squid catches of Korean and Japan, water temperatures at depth of 100 m, and night-visible images of fishing boats collected in the East (Japan) Sea from 1970 to 1999 were analyzed to examine the distribution and migration of the Japanese common squid, Todarodes pacificus, in the southwestern part of the East (Japan) Sea. The main fishing grounds detected from squid catch in each grid (0.5° latitude × 0.5° longitude) by Korean squid fishery and night-visible images provided by the Defense Meteorological Satellite Program (DMSP) Operational Linescan System (OLS) were situated in the southwestern part of the East (Japan) Sea. The distribution and migration route was illustrated from squid catch in each grid and DMSP OLS image. In years of high catches, the fishing grounds were situated mainly between Ulleung Island and the eastern coastal waters of Korea, while in years of low catches they were situated between Ulleung Island and the Yamato Bank in the central East (Japan) Sea. The center of fishing activity began moving northward from around the Korea/Tsushima strait to the northern boundary of the Tsushima Warm Current in March, reaching into the Yamato Bank in September, and then returning to the strait by February. The northward and southward migration routes differed; the northward migration route occurred closer to the mainland coast of Korea than the southward migration route did. This work suggests T. pacificus begin their northward migration almost 2 months earlier than previously suggested.     

Salmon testes meal as a functional feed additive in fish meal and plant protein‐based diets for rainbow trout (Oncorhynchus mykiss Walbaum) and Nile tilapia (Oreochromis niloticus L.) fry

Fishery processing by‐products are a large resource from which to produce fishmeal and other products for a variety of uses. In this study, testes meal (TM) produced from pink salmon processing by‐product was evaluated as a functional ingredient in aquafeeds. Nile tilapia and rainbow trout fry were fed five isonitrogenous and isoenergetic experimental diets for 4 and 9 weeks respectively. Two diets were fishmeal‐based (FM) and three were plant protein‐based (PP). Salmon TM was added to the FM and PP diets at 7% to replace 20% of fishmeal protein (FMTM and PPTM respectively). An additional control diet was prepared in which fishmeal was added to the PP diet to supply an equivalent amount of protein as supplied by TM (PPFM). Inclusion of TM in both the FM‐ and PP‐based diets resulted in higher final body weights, although differences were only significant between rainbow trout fed FM or FMTM diets. Similar differences were calculated for other indices of fish performance, e.g. specific growth rate, feed conversion ratio, protein efficiency ratio and protein retention efficiency. Feed intake was significantly higher for fish fed FMTM compared with FM in rainbow trout. For tilapia, final weights were numerically higher, but not significantly different for fish fed diets containing TM compared with non‐TM diets (FM vs. FMTM; PP vs. PPTM). Performance of trout or tilapia fed the PPFM diet did not increase compared with the PP diet. The results indicate that TM addition to both FM and PP diets increased feed intake and also increased metabolic efficiency, demonstrating that TM can be a functional ingredient in aquafeeds.     

Structure of the intratesticular duct system for sperm emission in the starspotted smooth-hound Mustelus manazo

We conducted histological observation of male germ cells and reproductive organs of the starspotted smooth-hound Mustelus manazo in Tokyo Bay to reveal any abnormality in male reproductive traits, as part of a study to elucidate the factors causing recent fluctuation in abundance of the population. Spermatogenesis proceeded in spermatocysts from the germinal zone in the ventral part of the testis to the degenerative zone in the dorsal part, where the spermatozoa were conveyed into the ciliated lumina of the attached terminal branches of the intratesticular ducts. The intratesticular ducts were classified from their terminal ends into branch, stem, and collecting tubules. The ducts formed in the germinal zone and grew as the spermatocysts developed. An opening formed through the wall of each of the most mature spermatocysts into a branch tubule; bundles of spermatozoa were evacuated through this opening into the branch and then the stem tubule and subsequently into the collecting tubules in the rete testis and the efferent duct connected to the epididymis. Spermatocysts that were unable to emit sperm because of failure of adhesion to the branch tubules were disorganized in situ, as were their spermatozoa. The collapsed spermatocysts seem to be cleared by hemophagocytosis with lymphocytes and leukocytes, which may have been recruited from the epigonal organ. There were no specific abnormalities in the spermatogenesis or the morphological structure of testes, which suggested that an abnormality of male reproductive traits was not the major cause of the recent fluctuation in the population abundance of this species. Details of the intratesticular duct system for sperm emission to the epididymis are the first findings in elasmobranchs worldwide.