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.     

Pigment deficiency correction in shrimp broodstock using Spirulina as a carotenoid source

In this study, an effort was made to cure the pigment deficiency syndrome (PDS) exhibited by pond‐reared Fenneropenaeus indicus (H.Milne Edwards) broodstock, using the blue‐green algae Spirulina as a carotenoid source in diet. The PDS symptoms included bleaching of ovary and reduced early larval performance. Biochemical analysis indicated reduced quantity of total carotenoids in egg and nauplii from the females with PDS. When PDS females were fed 30 g kg^?1Spirulina‐supplemented diet immediately after the appearance of symptoms, the egg and nauplii carotenoid values improved gradually and attained the pre‐PDS values after a minimum of 4 week period. Spirulina supplementation also improved significantly (P < 0.05) the spawn quality (hatch per spawn, nauplii per spawn, nauplii viability) and larval quality indices (P < 0.05). The egg and nauplii protein values exhibited a trend similar to carotenoids, confirming the influence of latter on protein stability previously reported by researchers. The study confirms the bioavailability of carotenoids from Spirulina to shrimp broodstock and recommends its regular inclusion in diet to preclude carotenoid deficiency‐related problems in shrimp hatcheries.     

Modulation of tissue α-tocopherol in African catfish, Clarias gariepinus (Burchell), fed oxidized oils, and the compensatory effect of supplemental dietary vitamin E

Juvenile African catfish, Clarias gariepinus (Burchell), of mean initial weight 15 g, were fed practical diets containing fresh or rancid oil (1:1 cod liver:corn oil) supplemented with either 20 or 100 mg all-rac-α-tocopheryl acetate per kg dry diet, at 0.03 × body weight per day for 8 weeks. After this time, catfish had grown by at least four times in body weight. Significant ( P < 0.05) inter-treatment differences in final body weight were noted. Clarias fed low-tocopherol: oxidized-oil diets performed least well with regard to growth, though elevated dietary vitamin E partially abrogated this effect. Growth of fish fed fresh-oil diets did not benefit from increased dietary α-tocopherol content. Muscle, liver, plasma, heart and spleen all responded significantly ( P < 0.05) to dietary vitamin E dose. Inclusion of oxidized oil in catfish diets decreased tissue α-tocopherol concentration. Hepatic α-tocopherol concentration (μg α-tocopherol per g liver) was observed to be lowered by 90% by the rancid oil diets. When fish previously fed fresh-oil diets were switched to oxidized: low-tocopherol diets, hepatic α-tocopherol concentration was significantly ( P < 0.05) lowered within 2 weeks. The results highlight the importance of dietary oil quality in modulating tissue α-tocopherol concentrations in African catfish.     

Gastric dilation and air sacculitis in farmed chinook salmon, Oncorhynchus tshawytscha (Walbaum)

A syndrome affecting cultured chinook salmon, Oncorhynchus tshawytscha (Walbaum), characterized by distended abdomens, gastric dilation, air sacculitis (GDAS), increased feed conversion rates and increased mortality has been recognized in New Zealand. Affected fish were most obvious in sea cages but were also present in fresh water. Mortality rates associated with this condition were highest in late summer and approached 6% per month. A dilated and flaccid stomach, without visible rugal folds containing copious oil, watery fluid or undigested feed was typical. Gastric mucosal ulceration or inflammation were not present. The air sacculitis consisted of a thickened, dilated bladder with a mixed mucosal inflammatory infiltrate and a luminal exudate associated with large numbers of morphologically diverse bacteria. Gastric dilation or air sacculitis occurred alone or together in the same fish. In a group of 20 subclinically affected fish with or without gastric dilation, there were no significant differences in weight, length, serum osmolality, sodium, total protein or packed cell volume. Twenty-three severely affected fish had significantly ( P  < 0.05) higher serum osmolality but similar sodium and total protein to that of clinically normal fish.     

Influence of high dietary α-tocopherol intakes on specific immune response, nonspecific resistance factors and disease resistance of healthy and aflatoxin B1-induced immunocompromised Indian major carp, Labeo rohita (Hamilton)

The effect of aflatoxin treatment and/or feeding of a high level of α‐tocopherol on immune response and disease resistance was investigated in Indian major carp, Labeo rohita. Group A served as a healthy control, group B was treated with aflatoxin, group C was fed a high level of α‐tocopherol whereas group D was exposed both to aflatoxin and a high level of dietary α‐tocopherol for 60 days. Aflatoxin B₁ (AFB₁) was injected once intraperitoneally into fish on the first day of the experiment (groups B & D). High levels of DL‐α‐tocopherol (1000 mg kg^–1 feed) were provided to healthy as well as AFB₁‐treated immunocompromised fish for 60 days (groups C & D). At the end of the experiment blood samples were assayed for changes in nonspecific immunity and humoral protein levels. Disease resistance against two common bacterial pathogens viz., Aeromonas hydrophila and Edwardsiella tarda were evaluated in all groups. Significant (P < 0.05) suppression of specific immunity as measured through haemagglutination (HA) titre against sheep red blood cells (SRBCs) as well as bacterial (formalin‐killed E. tarda) agglutination titre; nonspecific resistance factors viz., globulin level, serum bactericidal and lysozyme activities, neutrophil activities, and disease resistance against two bacterial pathogens only in aflatoxin‐treated fish with respect to the control group, clearly indicated the immunosuppressive nature of aflatoxin. Feeding of a high level of α‐tocopherol to AFB₁‐treated immunocompromised fish significantly (P < 0.05) raised specific immunity, nonspecific resistance factors and disease resistance capacity when compared with aflatoxin‐exposed fish. Disease resistance and enhancement of immune status through feeding of high levels of α‐tocopherol to healthy as well as AFB₁‐treated immunocompromised fish confirmed the potential of α‐tocopherol in carp feed for prevention of disease and for combating natural/environmental immunosuppressants.     

Sex reversal in the genus Oreochromis: optimization of feminization protocol

Abstract. High rate of feminization was achieved by oral administration of diethylstilbestrol (DES) and ethynyloestradiol (EES) to gonadally undifferentiated Oreochromis mossambicus (L.) and O. mossambicus × O. urolepis hornorum Trewavas all-male hybrid. All-female groups were produced at doses > 10mg/kg DES and >50mg/kg EES, when fry immediately after yolk-sac absorption were fed hormone-treated feed ad libitum. The critical period for feminization was about one week. Treatments longer than 10 days did not result in a higher proportion of females, and treatments starting later than one week after yolk-sac absorption were not effective. Optimal derived protocol was applied to O. aureus (Steindachner), resulting also in complete feminization.     

Volume regulation in glutathione-treated brook trout (Salvelinus fontinalis) erythrocytes

Brook trout erythrocytes that were washed with and suspended in Ringer's solution with reduced glutathione (1.0 mM) maintained steady state cell volume for up to 24h, while those without the thiol-protective agent steadily shrank. Changes in cell volume (measured as packed cell volume, PCV) were evoked by acidic media (Ringer's at pH 6.8), hypoosmotic solutions (or both) and intracellular K⁺ and Cl⁻ concentrations were monitored over 4h. Acid-swollen cells failed to volume regulate or release K⁺ but had significantly elevated intracellular Cl⁻ Osmotically-swollen cells at pH 7.8 but not at pH 6.8 underwent regulatory volume decrease (RVD) and returned to initial levels in 2h, accompanied by release of K⁺ and Cl⁻ In contrast, osmotically-shrunken cells did not show regulatory volume increase. The regulatory volume decrease and concomitant K⁺ release were dependent on Cl⁻ implying a direct or indirect coupling of K⁺ to Cl⁻ transport in volume regulation. RVD was partially blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0.1 mM), an anion exchange blocker, but was unaffected by amiloride (1.0 mM) which blocks Na⁺/H⁺ exchange. Amiloride and DIDS prevented the swelling response to low pH but had no effect on control cells, suggesting involvement of Na⁺/H⁺ and Cl⁻/HCO₃ ⁻ exchanges in acid-induced cell swelling. Quinine (1.0 mM) a known blocker of K⁺ channels, exacerbated the osmotically-induced swelling but had little effect on the subsequent RVD and release of KCl. The results suggest that low extracellular pH inhibits neutral C⁻-dependent K⁺ release and the resultant regulatory volume decrease in osmotically-swollen cells.     

Body shape variation within and among three species of darters (Perciformes: Percidae)

Abstract –  Using geometric morphometric (GM) techniques, we quantified intra and interspecific variation in female body shape using five collections each of Etheostoma caeruleum Storer, Etheostoma nigrum Rafinesque, and Etheostoma stigmaeum (Jordan). Nested multivariate analysis of variance showed significant variation in body shape among populations (Wilks' Λ = 0.0687, F _144,7585.1 = 19.35, P  < 0.0001), as well as among species (Wilks' Λ = 8.7 × 10⁻⁶, F _24,2 = 28.18, P  = 0.0348). Etheostoma caeruleum displayed greater body depth relative to both E. nigrum and E. stigmaeum , whereas E. nigrum displayed a compressed arrangement of mid-body landmarks relative to both E. stigmaeum and E. caeruleum . The broader implications of these findings highlight the value of geometric morphometrics as both an exploratory and analytical approach. Conclusions drawn from comparisons among moderately differentiated darter species in future studies of body size and shape are likely robust to intraspecific variation within species, and will permit more rigorous investigations into the ecomorphology of these benthic stream fishes.     

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.