Dina Zilberg

The effect of dietary protein level, water temperature and growth hormone administration on growth and metabolism in the common carp (Cyprinus carpio)☆

Abstract Two experiments were carried out to study fish growth and several other metabolic parameters under three main effects: dietary protein level (40% vs. 21%); water temperature (26 vs. 17°C), and intraperitoneal administration of carp growth hormone (cGH) (5 μg/g body weight/week vs. saline). Whereas both higher protein levels and elevated temperature promoted growth, the effect of cGH administration was highly dependent on protein level, the largest growth-promoting effect being found with low-protein diets. The dependence was particularly significant in the first experiment, in which the amount of feed provided was highest (5% vs. 2% of body weight/day). At the lower water temperature, fish in all treatments showed a significantly elevated hepatosomatic index. Protein level and cGH administration effects were unclear. The protein: fat ratio in the body contents was decreased by the high-protein diet and lower water temperature, whereas cGH had no effect. In contrast, the glycogen: fat ratio depended mainly on temperature—yielding higher ratios at the lower temperature, and not on cGH or protein levels. Carp GH administration decreased AcCoA carboxylase activity in the liver. Water temperature did not change the level of GH or T 3 in the plasma. Protein level did not influence GH but increased T 3 , whereas GH expression in the pituitary gland was downregulated by the administration of cGH and by the low-protein diet.

Soil nitrifying enrichments as biofilter starters in intensive recirculating saline water aquaculture

Abstract Intensive recirculating aquaculture relies on biofilters to sustain satisfactory water quality in the ponds. Establishment of new biofilters in aquaculture ponds without a start-up culture requires a long period of time and may therefore cause significant losses and environmental harm due to discharge of nitrogen-rich effluents. A laboratory scale setup (7-l aquaria with shrimp and fish) demonstrated that an external start-up nitrifying enrichment culture performed similarly to the natural bacterial population of an established pond biofilter, and superior to the performance of similar biofilters without a start-up culture (control). Ammonia concentration in the control treatment increased daily and reached 18 mg l −1 during a 14-day experiment, whereas in the treated aquaria, it averaged less than 2 mg l −1 . Fish growth and survival were similar in the treated aquaria (average growth of 0.45 g/14 days, and 95% survival) and significantly higher than in the control (average growth of 0.0 g/14 days, and 80% survival). The source for the enrichment cultures was soil samples collected from the region where the farm is situated. This approach may lead to the development of bacterial amendments (probiotic products) that can be used as start-up cultures for new operations or damaged filters, and potentially enhance nitrification in established filters. As the cultures are collected from soils, it is unlikely that they will be contaminated with fish disease-causing agents. This will improve water quality and consequently aquatic animal production.

Dried leaves of Rosmarinus officinalis as a treatment for streptococcosis in tilapia

Dietary application of dried Rosmarinus officinalis (rosemary) leaves as a treatment for streptococcal infection was studied in tilapia, Oreochromis sp. Feeding with dried rosemary leaves significantly reduced mortality following infection with Streptococcus iniae: 44% mortality in the group fed 8% rosemary, similar to oxytetracycline treatment (43% mortality), and significantly lower than the control (65%). Dietary administration of 16% rosemary significantly reduced mortality because of Streptococcus agalactiae infection in 44 g fish (62% and 76% in 16% rosemary and control, respectively), but not in a similar experiment conducted with 5.5 g fish. The antibacterial effect of rosemary on S. iniae was studied. Activity of rosemary cultivar Israel was reduced during the winter, but there was no significant change in cultivars Oranit and Star. Storage of powdered rosemary leaves at 50 degrees C resulted in fourfold and eightfold higher MIC(24 h) values after 3 and 4.5 months, respectively. Storage at -20 degrees C, 4 degrees C and 25 degrees C and autoclaving (120 degrees C) each resulted in a twofold increase in MIC(24 h). Repeated exposures of S. iniae to rosemary did not affect minimal inhibitory concentration, suggesting no development of resistance to rosemary.

Efficacy of garlic based treatments against monogenean parasites infecting the guppy (Poecilia reticulata (Peters))

Monogenean infections of commercially farmed fishes are responsible for significant economic losses. Garlic (Allium sativum) is a well-known spice which also possesses anti-microbial and anti-parasitical properties. The current work aimed to test the efficacy of garlic-based treatments against infection with monogenean sp. in the guppy (Poecilia reticulata). Clipped sections of tail fins of guppies heavily infected with Gyrodactylus turnbulli were exposed to aqueous garlic extract (7.5 to 30 mL L(-1)) and visually observed under a dissecting microscope. Results revealed that exposure to garlic caused detachment of parasite and cessation of movement indicating death. A positive correlation was seen between garlic concentration and time to detachment and death of parasites, which, at the highest concentration of 30 mL L(-1), occurred at 4.1 and 8.6 min, respectively. Bathing in aqueous garlic extract (7.5 and 12.5 mL L(-1)) was tested in guppies infected with G. turnbulli. Prior acute toxicity tests revealed the maximum tolerance levels of guppies to garlic extract to be 12.5 mL L(-1) for 1h. Bathing of infected fish in garlic extract (7.5 and 12.5 mL L(-1)) significantly (p<0.05) reduced infection prevalence and intensity as compared to the control. Oral treatments using dry garlic powder-supplemented diet were tested on guppies infected with G. turnbulli and Dactylogyrus sp. Fish were fed with food containing 10% and 20% dry garlic powder for 14 days. Groups fed with garlic supplemented diets showed significantly reduced (p<0.05) mean prevalence and mean intensity of parasites as compared to the control. Dietary application of garlic did not appear to affect palatability. Fresh crushed garlic was added at a level of 1 gL(-1) and applied as an indefinite bath for 14 days. This treatment was seen to significantly reduce (p<0.05) parasite prevalence and mean intensity as compared to the control. Histopathology revealed elevated muscular dystrophy in the 20% garlic-fed group, as compared to control. These findings demonstrate the potential of garlic as a natural alternative to currently used chemical treatments for monogenean sp. infection in the guppy.

Tetrahymena sp. infection in guppies, Poecilia reticulata Peters: parasite characterization and pathology of infected fish.

Tetrahymena sp. infection was diagnosed in guppies imported from Singapore. The parasite was isolated (Tet-NI) and optimally cultured in vitro in RM-9 medium. Cytological analyses [silver-staining and scanning electron microscopy (SEM)] revealed a pyriform-shaped, 64 x 41-microm holotrich ciliate without caudal cilium, containing a macro-nucleus (18.25 x 16.83 microm) and micro-nucleus (5.73 x 5.40 microm). Wet-mount examination and histological analyses of fish exposed to the parasite by co-habitation, immersion and infection by i.p. (intra-peritoneal) and i.m. (intra-muscular) injection revealed numerous ciliates on the skin, and in the gill and caudal fin blood vessels. Ciliates surrounded internal organs, the peri-orbital region of the eye, and were observed inside developing guppy embryos. Some muscle necrosis was associated with infection, but little or no inflammatory response. Immersion, co-habitation and i.m. injection caused relatively high infection rates and levels in the skin and tail, and lower infection in the gill blood vessels and internal organs; i.p. injection caused higher infection in the gill blood vessels and internal organs. Co-habited fish had relatively high infection levels in the hind-gut sub-mucosa. This is the first report of controlled systemic infection by Tetrahymena sp.

Protective immunization against Tetrahymena sp. infection in guppies (Poecilia reticulata)

Systemic tetrahymenosis constitutes a serious problem in guppy (Poecilia reticulata) production worldwide and no therapeutic solution is available for this disease. Three immunization trials were conducted, testing the effectiveness of different Tetrahymena preparations applied by intraperitoneal injection (IP) with or without Freunds complete adjuvant (FCA) and with or without booster dose. In trial 1, immunization with the pathogenic Tet-NI 6 lysate and live attenuated Tet-NI 1 did not provide significant protection from infection, although infection rates were significantly lower in the Tet-NI 6-immunized group than in controls. In trial 2, mortality in Tet-NI 6 + FCA-immunized fish was 10%, significantly lower than in all other treatment groups, including Tet-NI 6 lysate, live attenuated Tet-NI 1 and controls (77, 67 and 73%, respectively). In trial 3, the lowest mortality rates were obtained in the Tet-NI 6 + FCA + booster-immunized group (15%). These levels were lower but not significantly different from the non-boostered Tet-NI 6-immunized group (28%) and the groups immunized with Tet-NI 1, with and without booster (32 and 34%, respectively). Mortality in these four groups was significantly lower than in controls, including adjuvant- and PBS-injected groups (72 and 81%, respectively). Body homogenates of immunized fish immobilized Tetrahymena in-vitro, as compared to no or very little immobilization in controls. Lysozyme levels in the Tet-NI 6 + FCA + booster group were significantly higher than in all other treatments in trial 2 and controls in trial 3. There was no significant difference in anti-protease activity among the differently immunized fish. We conclude that immunization with Tetrahymena lysates in FCA confers a high degree of protection from infection, suggesting this preparation as a basis for vaccine development.

Efficacy of ginger-based treatments against infection with Gyrodactylus turnbulli in the guppy (Poecilia reticulata (Peters)).

Monogenean infections of commercially farmed fishes are responsible for significant economic losses and existing chemical therapeutants, often stressful to the fish, pose associated risks. As part of a recent trend to move towards the use of alternative, plant-based remedies for commonly occurring aquaculture-related diseases, the efficiency of ginger (Zingiber officinale) was investigated against the monogenean parasite Gyrodactylus turnbulli in the guppy. In vitro trials revealed the clear anti-parasitic effects of ginger. Ethanolic and aqueous extracts, prepared from freeze dried ginger, were tested. An increase in extract concentration was associated with reduced time to parasite immobilisation, with ethanolic extract being more efficient; at 75 and 200ppt aqueous ginger extract parasites died at 65.6±2.8 and 1.8±0.2min, respectively, whereas at 5 and 40ppt ethanolic extract parasites died at 26.1±0.7 and 4.9±0.3min, respectively. Bathing G. turnbulli-infected fish in ethanolic ginger extract (i.e. 5 and 7.5ppt for 90 and 30min, respectively) significantly reduced infection prevalence and intensity when compared to the water and ethanol controls. The higher concentration (i.e. 7.5ppt) proved as equally effective as Praziquantel, the conventionally used chemical treatment for gyrodactylosis, with the fish appearing to be completely cleared of the infection in both cases. Oral treatments of G. turnbulli-infected guppies with diets supplemented with 10 and 20% ginger powder proved to be ineffective in decreasing parasite load. These findings demonstrate that immersion in ginger extract offers an effective, alternative treatment against monogenean infection in fish.

Cysteine proteases and acid phosphatases contribute to Tetrahymena spp. pathogenicity in guppies, Poecilia reticulata.

Systemic tetrahymenosis caused by the protozoan parasite Tetrahymena spp. is a serious problem in guppy (Poecilia reticulata) farms worldwide. There is no therapeutic solution for the systemic form of this disease. Guppies severely infected with Tetrahymena spp. were imported by a commercial ornamental fish farm and brought to our laboratory. Tetrahymena sp. (Tet-NI) was isolated and in vitro cultured. Isolates maintained in culture for different time periods (as reflected by different numbers of passages in culture) were analyzed-Tet-NI 1, 4, 5 and 6, with Tet-NI 1 being cultured for the longest period (about 15 months, 54 passages) and Tet-NI 6 for the shortest (2.5 months, 10 passages). Controlled internal infection was successfully achieved by IP injection with most isolates, except for Tet-NI 1 which produced no infection. The isolate Tet-NI 6 induced the highest infection rates in internal organs (80% vs. 50% and 64% for Tet-NI 4 and 5, respectively) and mortality rates (67% vs. 20% and 27% for Tet-NI 4 and 5, respectively, and 6.7% for Tet-NI 1). The correlation between pathogenicity and Tetrahymena enzymatic activity was studied. Electrophoretic analyses revealed at least two bands of gelanolytic activity in Tet-NI 4 and 5, three bands in Tet-NI 6, and no activity in Tet-NI 1. Total inhibition of gelanolytic activity was observed after pretreatment of Tet-NI 6 with E-64, a highly selective cysteine protease inhibitor. Using hemoglobin as a substrate, Tet-NI 6 had two bands of proteolytic activity and no bands were observed in Tet-NI 1. A correlation was observed between pathogenicity and acid phosphatase activities (analyzed by commercial fluorescence kit) for Tet-NI 1 and Tet-NI 6.

Characterization of swim bladder non‐inflation (SBN) in angelfish, Pterophyllum scalare (Schultz), and the effect of exposure to methylene blue

Failure to inflate the swim bladder is regarded a major obstacle in the rearing of many fish species. We present a study of swim bladder non-inflation (SBN) in angelfish, Pterophyllum scalare. A normal developing primordial swim bladder was first discernable at the end of the first day post-hatch (p.h.) as a cluster of epithelial cells with a central lumen, surrounded by presumably mesenchymal cells. Initial inflation occurred on the fourth day p.h. Prior to inflation the swim bladder epithelium consisted of an outer squamous and inner columnar layer. Cells of the inner layer were filled at their basal region with an amorphous material, which disappeared upon inflation. A pneumatic duct was absent, and larvae presented no need to reach the water surface for inflation, suggesting that angelfish are pure physoclists. A model for the role of the amorphous material in normal initial inflation is proposed. Abnormal swim bladders were apparent from the fourth day p.h., and methylene blue (MB) at a concentration of 5 ppm significantly increased the prevalence of SBN. Histologically, abnormal swim bladders in larvae hatched in 5 ppm MB could not be distinguished from those in fish raised under routine conditions (0.5 ppm MB). We suggest that MB may have a teratogenic effect in angelfish.

Comparative study of infection with Tetrahymena of different ornamental fish species.

Tetrahymena is a ciliated protozoan that can infect a wide range of fish species, although it is most commonly reported in guppies (Poecilia reticulata). The aim of this study was to compare the susceptibility to infection with Tetrahymena of five different ornamental fish species from two different super orders. The species examined were platy (Xiphophorus), molly (Poecilia sphenops) and angelfish (Pterophyllum scalare) of the Acanthopterygii super order (which also includes guppies) and goldfish (Carassius auratus auratus) and koi carp (Cyprinus carpio) of the Ostariophysi super order. These two super orders are phylogenetically distant from each other. Infection with Tetrahymena resulted in parasite invasion of internal organs, skin and muscle in all fish species. A relatively strong inflammatory response was observed in infected goldfish and koi, with negligible response in fish species of the Acanthopterygii super order. Guppies were the most susceptible to Tetrahymena infection, exhibiting a mortality rate of 87% and 100% in two separate experiments. A high mortality rate was also observed in platy (77%), while that of molly and angelfish was significantly lower (23% and 33%, respectively). Goldfish and koi carp were less susceptible to infection compared with guppies (24% and 59% mortality, respectively). Immunization studies revealed that the Tetrahymena are immunogenic, since infection of koi carp increased their Tetrahymena immobilization response by approximately three-fold at 3 weeks post infection, while immunization with Tetrahymena plus adjuvant increased their immobilization response by approximately 30-fold.