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.     

Dietary phosphorus requirement of juvenile Atlantic salmon, Salmo salar L.

The objective of this study was to determine the dietary phosphorus (P) requirement of juvenile Atlantic salmon, Salmon salar L. Triplicate groups of fish (mean initial weight 1.4 g) were fed semipurified, casein-gelatine-based diets containing one of five levels of P (4, 8, 10, 15 and 25 g kg⁻¹) from Ca(H₂PO₄)₂·H₂O, or a commercial feed (17 g kg⁻¹ P) for 9 weeks. Weight gains did not differ significantly among treatment groups fed the experimental diets but were slightly less than gains in fish fed the commercial feed. Feed efficiency (wet weight gain/dry feed consumed) was similar in all groups, averaging 1.45. Availability of dietary P, estimated from apparent retention and apparent digestibility, was 86%. Whole-body P concentrations declined in fish fed diets containing less than 10 g kg⁻¹ P. Fitting a logistic curve to dietary P vs. whole-body P concentrations indicated that a minimum of 11 g kg⁻¹ dietary P (9 g kg⁻¹ digestible P) was required by juvenile Atlantic salmon to maintain whole-body P concentrations at initial levels. Calculation of a dietary requirement using a simple factorial model which incorporated measurements of P availability, feed efficiency and normal whole-body P concentration indicated that the dietary requirement was approximately 10 g kg⁻¹. The dietary requirement established in this study (10–11 g kg⁻¹) is higher than previously reported for Atlantic salmon or other fishes. Possible reasons for the wide range of reported dietary P requirements in fishes are discussed.     

Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.     

Melanomacrophages in three species of free-ranging sharks from the northwestern Atlantic, the blue shark Prionacae glauca (L.), the shortfin mako, Isurus oxyrhinchus Rafinesque, and the thresher, Alopias vulpinus (Bonnaterre)

The melanomacrophage aggregates or cells (MMC) are commonly used as biomarkers of exposure to pollution in fish, albeit their numbers and morphological characteristics can be influenced not only by environmental toxins but also by a range of physiological parameters and pathological conditions. Accordingly, before we can use MMC as biomarkers in any fish species, their normal, 'background' characteristics have to be established in apparently healthy fish. The knowledge of MMC in sharks is minimal. The aim of this study was to characterize MMC from 51 free-ranging, large pelagic sharks from the northwestern Atlantic, including shortfin mako, Isurus oxyrhinchus , thresher, Alopias vulpinus and blue shark, Prionacae glauca . The rationale of this study was twofold. First, because, sharks have life histories predisposing them to the accumulation of environmental toxins they constitute good sentinel species of the health of the global marine ecosystem. Second, because many shark populations are in decline or threatened by extinction, we need to expand our understanding of their health status in order to develop appropriate protective measures. All sharks were collected by sports fishing gear between June and August in 2007. Their health condition was assessed by necropsy, morphometrics, and by microscopic examination of gonads, livers, spleens and kidneys. Routine, haematoxylin and eosin and/or Pearl's reagent-stained paraffin embedded sections were studied by light microscopy. Our results provide the first data on the morphometric and morphological characteristics of MMC in viscera of apparently healthy free-ranging sharks from the northwestern Atlantic.     

Effect of dietary protein/carbohydrate ratio on activities of hepatic enzymes involved in the amino acid metabolism of Nile tilapia, Oreochromis niloticus (L.)

This study investigated the influence of different dietary protein/carbohydrate ratios on activities of enzymes involved in the amino acid metabolism as well as on growth performance and body composition of Nile tilapia (Oreo- chromis niloticus). Fish were fed semi-synthetic diets differing in their protein/carbohydrate ratio. The diets were formulated to be isolipidic and isoenergetic (on the basis of metabolizable energy). Fish were reared individually. After a prefeeding phase, tilapia were randomly assigned to three groups and were fed the experimental diets at a relatively low level. The study lasted 9 weeks. Proximate composition, gross energy content, and activities of aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) in the liver of fish were determined. While the mean growth rate and feed conversion efficiency did not differ significantly among the feeding groups, the body composition showed significant differences. The dietary protein/carbohydrate ratio had no significant effect on the protein and energy gain, but the lipid gain was significantly higher in tilapia fed the low-protein diet. The activities of ASAT and ALAT per gram of liver and per gram of soluble protein were significantly increased in fish fed the high-protein diet. There was a significant positive correlation between the activities of ASAT and ALAT. The activities of both enzymes in the liver were significantly higher in fish with a higher protein gain. Measuring the activities of enzymes involved in the amino acid metabolism can provide more information about the metabolic utilized dietary protein.     

Compensatory growth in Atlantic halibut: Effect of starvation and subsequent feeding on growth, maturation, feed utilization and flesh quality

A group of juvenile Atlantic halibut (mean, SD, 199.5 g, 44.7) was split into two experimental groups: one group was fed in excess twice a day and later five days a week (i.e. Control group), and the other group was starved for 5 weeks and then subsequently re-fed for 10 weeks (i.e. 5/10 starvation/re-fed group). This treatment was repeated for three years until the fish were harvested (mean final weight, 4.4 kg). A size +specific compensatory response was seen; partial compensation was observed in halibut weighing less than 500 g whereas full recovery growth was seen in halibut over 2 kg. Food consumption and feed conversion efficiency was measured during two full 5/10 starvation/re-fed cycles. The Control group consumed 86% more food in the measurement period, whereas the FCE 40% higher in the 5/10 starvation/re-fed group (FCE = 0.95) compared to the Control group (FCE = 0.68). In males lower gonadosomatic index (2.2 vs. 3.0) and plasma 11-ketotestosterone (0.22 vs. 1.26 ng ml^− 1) levels in the 5/10 starvation/re-fed group were observed during late winter and spring of 2006 (age 3 years) indicating higher age at 1st maturity. Starvation had a significant effect for improving the quality as starved fish had higher muscle pH (6.4 vs. 6.2), less gaping (0.1 vs. 0.3) and harder texture (61.4 vs. 57.6 N). Overall, the rearing on a repeated 5/10 starvation/re-fed regime for 3 years led to full growth compensation, higher feed conversion efficiency, lower male maturation and improved flesh quality.     

Relative potencies of natural estrogens on vitellogenin and choriogenin levels in the Indian freshwater spotted snakehead, <Emphasis Type="Italic">Channa punctata</Emphasis>: in vivo and in vitro studies

The relative efficacies of three natural estrogens viz., estrone (E₁), estradiol-17β (E₂) and estriol (E₃) to induce synthesis of vitellogenin (Vg) and choriogenin (Chg) were assessed in primary hepatocyte cultures of the Indian freshwater spotted snakehead, Channa punctata. Hepatocytes were isolated from the spotted snakehead liver by a non-enzymatic protocol. Optimum culture conditions were standardized for ensuring their viability and functioning. Isolated hepatocytes were cultured for 48 h for monolayer formation and then exposed to various concentrations (0.001–10 μM) of the three estrogens. Competitive homologous ELISAs, developed and validated for spotted snakehead Vg and Chg were employed to determine the amounts of these two proteins secreted into the culture medium after 48 h of incubation. The results reveal that although all the three estrogens were effective in inducing the production of Vg and Chg in a dose-dependent manner, there were differences in their relative potencies. Of three estrogens, E₁ was the least potent and could induce synthesis of Vg and Chg only at a minimum concentration of 0.5 μM; whereas significant levels of both the proteins were quantified in culture medium by exposing the hepatocytes to E₂ or E₃ even at a concentration of 0.001 μM. All three estrogens were effective in inducing synthesis of Vg and Chg in vivo also. These results suggest the possibility of employing the above in vitro experimental design to monitor the presence of estrogens/estrogen-like chemicals in natural waters, which could interfere with the estrogen receptor system of fish. This study further points to the possibility of using Chg, in addition to Vg, as a parameter for screening various chemicals for their estrogenic activity.     

Description and phylogeny of Ceratomyxa anko sp. n. and Zschokkella lophii sp. n. from the Japanese anglerfish, Lophius litulon (Jordan)

Two new species of myxozoans from the Japanese anglerfish, Lophius litulon, are described using myxospore morphology and small subunit rDNA sequences. Ceratomyxa anko sp. n. is a parasite of the gall bladder and had a prevalence of 57%. Mature spores of C. anko sp. n. are arcuate to crescent shaped with valves tapering to rounded tips. A prominent sutural line runs centrally between the round adjacent polar capsules containing the polar filament coiled two to three times. Spore measurements: length 10.8 (9.7-11.9) microm, width 41.9 (36.9-47.2) microm, polar capsule diameter 4.6 (4.1-5.3) microm. Ceratomyxa anko sp. n. can be distinguished from other Ceratomyxa spp. due to its spore dimensions and shape. Zschokkella lophii sp. n. is a parasite of the urinary bladder and had a prevalence of 70%. Mature spores are ellipsoidal to semicircular with bluntly pointed ends. The sutural line is curved or sinuous and the valves have no discernable surface ornamentation. Two almost spherical polar capsules are located separately in the ends of the spore, opening in almost opposite directions and contain the polar filament with five coils. Spore measurements: length 20.1 (16.8-24.0) microm, width 14.9 (12.7-16.8) microm, polar capsule diameter 5.1 (3.6-5.8) microm. Zschokkella lophii sp. n. can be distinguished from other Zschokkella spp. due to the terminal opening of the polar capsules within the spores and the site of infection within the host fish. In the phylogenetic analyses, C. anko sp. n. grouped with other members of the same genus forming a monophyly. Zschokkella lophii sp. n. forms a discrete clade with another Zschokkella sp. that infects the urinary bladder of marine fish. This grouping forms a sister clade to one containing members of the genus Parvicapsula, all of which are parasites of the urinary system in marine fish.     

Biochemical composition and quality of captive-spawned cobia Rachycentron canadum eggs

Interest in the commercial production of cobia Rachycentron canadum continues to rise as additional insight is gained into the hardy and fast growing nature of this species. However, research regarding the biochemical composition of captive-spawned eggs and egg and larval quality remains scarce. Such data is essential as a common bottleneck to production is a steady supply of fingerlings for grow-out. This study quantified the biochemical composition and quality of cobia eggs produced over 2 spawning seasons by broodstock on a traditional ‘trash fish’ diet which is commonly fed to tank spawning cobia. Throughout the study, batch fecundity, proportion of floating eggs and percent hatch averaged > 1 million eggs,  0.8 and 70%, respectively. Batch fecundity was significantly higher during the second spawning season as a result of the increased size of the females which weighed 18/22 kg and 22/26 kg at the beginning of each season. A positive correlation was found between the proportion of floating eggs and hatch rate for both spawning seasons. No correlations were found between egg composition (total lipid (30.0 ± 1.1% dry wt), protein (25.4 ± 2.2% dry wt), carbohydrate (2.4 ± 0.3% dry wt), vitamin E (10.2 ± 0.6 μg/g wet wt) or dry weight (119.1 ± 5.5 μg/egg)) and egg quality (proportion of floating eggs, hatch rate, larval growth and larval survival). Further, no differences in egg composition were noted between seasons or over the course of each season. The fatty acid composition of cobia eggs varied between seasons possibly due to changes in the quality of frozen feed (fish, shrimp, squid) given to the broodstock. The only correlation between the fatty acid profile and egg quality was a decrease in the proportion of floating eggs as the total amount of n-3 highly unsaturated fatty acids increased. No relationship between egg quality and amino acid content was noted with the most prominent amino acids being glutamate, leucine, alanine, proline, lysine and aspartate nor were any differences detected between spawning seasons.