Validation,use, and disadvantages of enzyme-linked immunosorbent assay kits for detection of cortisol in channel catfish,largemouth bass,red pacu,and golden shiners

Measurement of cortisol response is an important tool to asses stress in fisheries research. Radioimmunoassay (RIA) is a common method for the measure of cortisol in fish. Use of enzyme-linked immunosorbent assays (ELISA) to detect cortisol would eliminate health hazards, costs of handling radioisotopes, and the short stability time associated with RIA. Enzyme-linked immunosorbent assays have been used for the determination of cortisol in several fish species. However, the ELISA method of cortisol determination in fish lacks proper validation testing. We conducted validation procedures for multiple commercial cortisol ELISA kits and compared the results to RIA. The assays were tested for four species: (1) channel catfish Ictalurus punctatus, (2) largemouth bass Micropterus salmoides, (3) red pacu Piaractus brachypomus, and (4) golden shiners Notemigonus crysoleucas. We evaluated the ELISA methods against RIA, and determined that at least one kit is suitable (accuracy: mean recovery of spiked samples, 102.8%; reproducibility: interassay coefficient of variation < 10.5% for all species; precision: intra-assay coefficient of variation < 16.8% for all species; linearity: R ² > 0.96 for all species) for the measurement of cortisol response in fish and comparative determination of stress. All of the ELISA assay results varied by more than 10% from the cortisol concentrations detected by the RIA. The high variability of the kit results indicates that commercial ELISA kits could be utilized for qualitative determination of cortisol in fish, but should be fully validated in each laboratory for each species before being used for research.     

The use of a real-time PCR primer/probe set to observe infectivity of Yersinia ruckeri in Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and steelhead trout, Oncorhynchus mykiss (Walbaum)

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a common pathogen affecting aquaculture facilities and implicated in large losses of cultured fish. Fisheries scientists continue to gain a greater understanding of the disease and the pathogen by investigating methods of identification and pre- and post-infection treatment. In this study, a real-time PCR probe set for Y. ruckeri was developed to detect daily changes in the bacterial load during pathogen challenges. Two species of fish, Chinook salmon, Oncorhynchus tshawytscha, and steelhead trout, Oncorhynchus mykiss, were exposed to two strains of Y. ruckeri (Hag and SC) during bath challenges. A subset of fish was killed daily for 14 days, and the kidney tissue was biopsied to enumerate copies of pathogen DNA per gram of tissue. While Chinook exposed to either the Hag or SC strains exhibited similar pathogen loads, those exposed to the Hag strain displayed higher mortality (～66%) than fish exposed to the SC strain (～24% mortality). Steelhead exposed to the Hag strain exhibited a greater pathogen load and higher mortality (～42%) than those exposed to the SC strain (<1% mortality). Steelhead challenged with either strain showed lower pathogen loads than Chinook. The study illustrates the efficacy of the probe set to enumerate Y. ruckeri bacterial growth in the kidneys of fish. Also, strains of Y. ruckeri display species-specific growth patterns that result in differential mortality and pathogen load.     

Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg)

An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN.     

Movements and behaviors of swordfish in the Atlantic and Pacific Oceans examined using pop‐up satellite archival tags

Swordfish are highly specialized top‐level predators that have been challenging to study. In this paper, data from 31 pop‐up satellite archival tags attached to swordfish from (i) the eastern Pacific, (ii) central Pacific, and (iii) western North Atlantic‐Caribbean were analyzed. Common across locations was a pronounced diel vertical pattern with daytime hours spent primarily below the thermocline and nighttime hours spent in warmer waters, close to the surface. One exception to this pattern was periodic daytime basking events which were most common in cooler waters off California. Maximum daytime depths were significantly correlated with light penetration as measured by the diffuse attenuation coefficient at 490 nm. Temperature did not appear to influence daytime depths, and swordfish tolerated both extremely low temperatures (4°C) and rapid and dramatic temperature changes (>20°C). Temperature did appear to influence the nighttime depths in the Pacific where fish typically remained in the surface mixed layer. In contrast, in the warm tropical Atlantic this was not the case, and nighttime depths were much deeper. In all areas, nighttime depth increased around the full moon. Given the parallels between the vertical movement patterns of swordfish and those of the deep sound scattering layer we suggest that swordfish vertical distribution patterns, especially during daytime, are influenced largely by resource availability. At night, when swordfish are typically targeted by fisheries, both ambient light and temperature influence movements. Understanding vertical movement patterns of swordfish can help evaluate gear vulnerability, improve population assessments, and potentially reduce fisheries bycatch.     

Opto-electronic counters for postlarvae and juveniles of Machrobrachium rosenbergii (de Man); design and tests of Type A counter

Postlarvae or juveniles of Macrobrachium rosenbergii produced in Mauritius are sold at unit price to prawn farmers and are counted individually by hand. This process is time consuming, labour intensive, subject to error and may stress the juveniles; safe rapid electronic counting would be highly advantageous.The design of the hydraulic component of a successful counter prototype, Type A, is described. The counter has 10 parallel channels through which juveniles are carried in a water current. The detection of juveniles passing through these channels can be done photo-electrically. The counter accepts juveniles up to 15 mm in length (eye orbit to tip telson) and has the significant advantage that it can be constructed using only simple hand tools.Survival and growth rate tests, and microscopic examination for damage to appendages of juveniles were done on machine counted and hand counted juveniles. There was no reduction of growth or survival caused by passage through the counter by comparison with the hand counting method, and it is concluded that the counter is not detrimental to juvenile prawns.About 70% of all juveniles tested showed highly variable amounts of damage. The great majority of this damage appears to originate in the hatchery tanks, prior to the experimental treatments. Only in the case of antennules could the use of the counter be shown to increase damage significantly, i.e. from 25% to 38%, but this has no detectable effect on subsequent survival or growth.This appears to be the first counter made specifically for counting macroscopic crustacean juveniles, and via suitable scaling, adaptation to counting other species of crustaceans, fish larvae, etc., should be possible.     

Diurnal variations in hypothalamic monoamine levels in the teleostChanna punctatus (bloch) in response to melatonin under two photothermal conditions

Hypothalamic dopamine (DA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT) levels exhibited marked day-night variations under ambient photoperiod and temperature (12L12D; 17±1°C) with peak values at mid-light phase. The 16L8D; 22±1°C treatment reversed the diurnal rhythm of 5-HT, but not that of DA and NA. However, there was an overall increase in the levels of the catecholamines on exposure to the long photoperiod and high temperature. The administration of melatonin in the fish held on 16L8D; 22±1°C regime restored the 5-HT rhythm to that of the 12L12D; 17±1°C control group, but with elevated mid-photophase value. However, there was no effect of the indole treatment on the 5-HT rhythm under the 12L12D; 17±1°C regime. Melatonin causes a significant reduction of NA level in both the groups, while DA level did not change in either group.     

Application of Azotobacter enhances pond productivity and fish biomass in still water ponds

Two strains (derepressed-nitrogen fixing, Mac-27 and phosphate solubilizing, PS-21) of Azotobacter chroococcum were inoculated in fish culture ponds, singly and in combination with inorganic fertilizers (urea, single superphosphate–SSP). Physico-chemical parameters of pond waters, plankton production and fish biomass were studied. Inoculation of A. chroococcum (Mac-27) enhanced nitrogenase activity and rate of nitrogen fixation. A slight reduction in nitrogen fixation and nitrogenase activity was noticed when urea at 96 kg ha–1 y–1 was mixed with the biofertilizer (Mac-27). Inoculation of PS-21 enhanced phosphate solubilization, but Kjeldahl-nitrogen concentration values remained low in comparison with controls. On the other hand, inoculation of Azotobacter (either strain) enhanced the accumulation of ammonium-N, nitrite-N and nitrate-N. A significant (p < 0.05) reduction in dissolved oxygen (DO) concentration also took place when Azotobacter (both Mac-27 and PS-21) was inoculated in fish ponds. However, when used along with inorganic fertilizers, the reduction was not significant. The pH values were only slightly lowered when the phosphate-solubilizing strain (PS-21) of Azotobacter was inoculated. Inoculation of biofertilizer enhanced plankton production, net primary productivity and fish biomass. However, highest values in most of these parameters were noticed only in ponds that were treated with the higher doses of inorganic fertilizers (urea 192 kg and SSP 1500 kg ha–1 y–1). © Rapid Science Ltd. 1998     

The utilization of clam waste meal as a protein source for rainbow trout Salmo gairdneri

Solid waste recovered from clam processing wash water was evaluated for its utilization as a replacement protein for fish meal. Three diets, diets 1 and 2 formulated to simulate the Oregon Moist Pellet (OMP) and diet 3 (Rangen), were fed to rainbow trout (Salmo gairdneri) for eight weeks. The effects of the clam waste meal on fish growth, food conversion, carcass composition and whole body fatty acid content were determined. The clam waste diet supported maximum growth and food conversion. Carcass analysis of moisture, protein and lipid contents were similar among the moist feeds. The dietary levels of 20:5w3 + 22:6w3 and total polyunsaturated fatty acids (PUFAs) were significantly higher in the clam waste diet pellet.     

The effect of Eimeria sp. infection on the condition of blue whiting, Micromesistius poutassou (Risso)

Abstract. Eimeria sp. infection of blue whiting, Micromesistius poutassou (Risso), taken to the north and west of Scotland, was investigated with particular reference to the effect on host condition. Eimeria sp. is a parasite of the liver parenchyma and causes extensive lesions. Juvenile blue whiting were not found to be infected before the late 1-group stage, but all 308 adult (3- to 17-group) fish examined were infected. Condition factor ( k = 100 w/l³ ) showed a statistically significant decrease with increasing intensity of infection. Average value of liver weight, expressed as a percentage of total body weight, also decreased with increasing intensity of infection. It is concluded that Eimeria sp. infection is a major factor contributing to loss of condition in blue whiting from the study area. The loss of weight in fish with moderate-to-heavy infections was estimated at from 6 to 10%; this figure does not take account of mortalities resulting from Eimeria infection nor does it take account of the most heavily infected fish which could not be included in the analysis because sufficiently accurate estimates of the large numbers of oocysts present were not made.