Ian Bricknell

Changes in the gut-associated microflora during the development of Atlantic halibut (Hippoglossus hippoglossus L.) larvae in three British hatcheries

The bacterial flora of reared Atlantic halibut eggs, larvae, juveniles and adults from three different UK halibut hatcheries was monitored using a combination of traditional biochemical tests, the BIOLOG GN bacterial identification system, PCR-RFLP of 16S rRNA genes and partial 16S rDNA gene analysis. A gut microflora was detected towards the beginning of the non-feeding yolk-sac stage, with a substantial increase as the larvae started to feed. Bacteria isolated from non-feeding yolk-sac larvae were predominantly non-fermentative Gram-negative rods, in particular Pseudoalteromonas species; by contrast, the presumptive gut microflora of first-feeding larvae was generally dominated by members of the Vibrio genus. There was evidence of a bacterial succession after the larvae started to feed. Initially, first-feeding halibut reared on enriched Artemia were generally colonised by live food-associated bacteria, particularly Vibrio splendidus and V. alginolyticus-type organisms. Genetically similar V. splendidus isolates were recovered from enriched Artemia and the guts of first-feeding halibut larvae in all three hatcheries. However, only a selected proportion of the Artemia-associated bacteria appeared able to establish themselves within the guts of larval halibut. First-feeding halibut larvae were sometimes colonised by opportunistic bacteria. The gut floras of two individual batches of halibut larvae were briefly dominated by a Pseudoalteromonas and a Photobacterium phosphoreum isolate, respectively. Genetically-similar V. salmonicida-type organisms were present in larvae sampled from all three hatcheries, both from copepod and Artemia-fed fish. The guts of the adult halibut analysed had a culturable flora dominated by Photo. phosphoreum.

Survival and replication of Piscirickettsia salmonis in rainbow trout head kidney macrophages

Piscirickettsia salmonis is pathogenic for a variety of cultured marine fish species worldwide. The organism has been observed within host macrophages in natural disease outbreaks among coho salmon and European sea bass. In vitro studies, incorporating transmission electron microscopy (TEM) and ferritin loading of lysosomes, have confirmed that P. salmonis is capable of surviving and replicating in rainbow trout macrophages. Certain features of this intracellular survival underline its difference to other intracellular pathogens and suggest that a novel combination of defence mechanisms may be involved. Escape into the macrophage cytoplasm is not used as a means to avoid phago-lysosomal fusion and the organism remains at least partly enclosed within a vacuole membrane. While the piscirickettsial vacuole is often incomplete, survival and replication appear to require occupation of a complete, tightly-apposed, vacuolar membrane which does not fuse with lysosomes. Unlike some mammalian rickettsiae, actin-based motility (ABM) is not used as a means of intercellular spread. It is postulated that the presence of numerous small vesicles within vacuoles, and at gaps in the vacuolar membrane, may result from the blebbing of the piscirickettsial outer membrane seen early in the infection.

Susceptibility of Atlantic halibut, Hippoglossus hippoglossus (L.) to infection with typical and atypical Aeromonas salmonicida

Abstract Halibut, Hippoglossus hippoglossus (L.) and Atlantic salmon, Salmo salar (L.) were challenged with virulent typical or atypical strains of Aeromonas salmonicida by both injection and bath models. The groups were injected intraperitoneally with 100 μl of logarithmically decreasing dilutions of A. salmonicida (range 108–103 cells/fish) or bathed in 105 CFUs/ml of A. salmonicida for 24 h. Halibut were significantly more resistant to infection, compared to Atlantic salmon, with 106 and 107 typical and atypical A. salmonicida cells/halibut being the minimum lethal dose. No halibut died in the bath challenge although approximately 80% of salmon died by this challenge method. All animals that died during the challenge were positive for A. salmonicida on culture A. salmonicida was cultured from internal organs of approximately 60% of surviving Atlantic salmon while only 2.3% of the surviving halibut were culture positive, but only from the intestinal lumen. All surviving salmon but none of the surviving halibut, showed histological evidence of infection with A. salmonicida. Stress tests of both halibut and Atlantic salmon showed that recovered salmon still had a high carrier rate of A. salmonicida while none of the surviving halibut were carrier test positive. A significant rise in agglutinating antibody titre was observed in surviving Atlantic salmon. However, no rise in antibody titre was observed in the surviving halibut, suggesting that the processing of the bacteria during an acute infection may be fundamentally different between these two species.

Effects of different water treatment methods and antibiotic addition on larval survival and gut microflora development in Atlantic halibut (Hippoglossus hippoglossus L.) yolk-sac larvae

Abstract The survival of Atlantic halibut ( Hippoglossus hippoglossus ) yolk-sac larvae and the development of their gut bacterial flora were compared in pilot-commercial scale rearing trials under different conditions. Four different treatments were tested; water recirculation using non-disinfected embryos (RN); water recirculation using disinfected embryos (RS); flow-to-waste water with disinfected embryos (F); flow-to-waste water with disinfected embryos and addition of antibiotics, oxytetracycline, oxolinic acid and amoxycillin at 20 mg l −1 (FA). Mean survival was greatest in the FA group (53%), which was significantly higher ( p 71% morphologically normal larvae in all treatments. Addition of antibiotics appeared to prevent the colonisation of yolk-sac larvae by Marine Agar (MA)-culturable bacteria ( 2 CFU/larva for group F). Higher levels of bacteria were recovered from both groups of larvae reared in recycled water (5.8×10 3 CFU/larva for group RN and 7.79×10 3 for group RS), indicating that bacterial abundance is not, in itself, a determinant of survival. Differences in the types of bacteria that colonised the halibut yolk-sac larvae under the different rearing regimes were described by characterization of the isolates. There appeared to be a relationship between larval survival rate and the presence, or absence, of certain phenotypes. In particular, the gut microflora in the poorest performing group (group F) was dominated by isolates biochemically and morphologically distinct from the predominant Pseudoalteromonas species isolated from the better performing recycled water groups. This lends weight to the hypothesis that certain types of bacteria may act as opportunistic pathogens in yolk-sac incubators while others are benign.

Experimental Transmission of Infectious Pancreatic Necrosis Virus from the Blue Mussel, Mytilus edulis, to Cohabitating Atlantic Salmon (Salmo salar) Smolts

ABSTRACT Integrated multitrophic aquaculture (IMTA) reduces the environmental impacts of commercial aquaculture systems by combining the cultivation of fed species with extractive species. Shellfish play a critical role in IMTA systems by filter-feeding particulate-bound organic nutrients. As bioaccumulating organisms, shellfish may also increase disease risk on farms by serving as reservoirs for important finfish pathogens such as infectious pancreatic necrosis virus (IPNV). The ability of the blue mussel (Mytilus edulis) to bioaccumulate and transmit IPNV to naive Atlantic salmon (Salmo salar) smolts was investigated. To determine the ability of mussels to filter and accumulate viable IPNV, mussels were held in water containing log 4.6 50% tissue culture infective dose(s) (TCID50) of the West Buxton strain of IPNV ml−1. Viable IPNV was detected in the digestive glands (DGs) of IPNV-exposed mussels as early as 2 h postexposure. The viral load in mussel DG tissue significantly increased with time and reached log 5.35 ± 0.25 TCID50 g of DG tissue−1 after 120 h of exposure. IPNV titers never reached levels that were significantly greater than that in the water. Viable IPNV was detected in mussel feces out to 7 days postdepuration, and the virus persisted in DG tissues for at least 18 days of depuration. To determine whether IPNV can be transmitted from mussels to Atlantic salmon, IPNV-exposed mussels were cohabitated with naive Atlantic salmon smolts. Transmission of IPNV did occur from mussels to smolts at a low frequency. The results demonstrate that a nonenveloped virus, such as IPNV, can accumulate in mussels and be transferred to naive fish.

Fat cell necrosis syndrome in Atlantic halibut, Hippoglossus hippoglossus L.

Abstract A progressive degenerative condition of the dorsal sub-dermal fat deposits of farmed Atlantic halibut is reported, nominally termed fat cell necrosis syndrome (FCNS). The disease does not appear to have an infectious or malignant aetiology, but may be related to an imbalance between dietary oxidants and antioxidants combined with an exposure to sunlight. The gross lesions and histopathology are described.

Factors affecting post-capture survivability of lobster Homarus americanus.

Technological advances in gear and fishing practices have driven the global expansion of the American lobster live seafood market. These changes have had a positive effect on the lobster industry by increasing capture efficiency. However, it is unknown what effect these improved methods will have on the post-capture fitness and survival of lobsters. This project utilized a repeated measures design to compare the physiological changes that occur in lobsters over time as the result of differences in depth, hauling rate, and storage methodology. The results indicate that lobsters destined for long distance transport or temporary storage in pounds undergo physiological disturbance as part of the capture process. These changes are significant over time for total hemocyte counts, crustacean hyperglycemic hormone, L-lactate, ammonia, and glucose. Repeated measures multivariate analysis of variance (MANOVA) for glucose indicates a significant interaction between depth and storage methodology over time for non-survivors. A Gram-negative bacterium, Photobacterium indicum, was identified in pure culture from hemolymph samples of 100% of weak lobsters. Histopathology revealed the presence of Gram-negative bacteria throughout the tissues with evidence of antemortem edema and necrosis suggestive of septicemia. On the basis of these findings, we recommend to the lobster industry that if a reduction in depth and hauling rate is not economically feasible, fishermen should take particular care in handling lobsters and provide them with a recovery period in recirculating seawater prior to land transport. The ecological role of P. indicum is not fully defined at this time. However, it may be an emerging opportunistic pathogen of stressed lobsters. Judicious preemptive antibiotic therapy may be necessary to reduce mortality in susceptible lobsters destined for high-density holding facilities.

Recovery from a near-lethal exposure to ultraviolet-C radiation in a scleractinian coral

Hermatypic (reef building) corals live in an environment characterized by high ambient levels of photosynthetically active radiation (PAR) and ultraviolet radiation (UVR). Photoadaptive mechanisms have evolved to protect the sensitive cell structures of the host coral and their photosynthetic, endosymbiotic zooxanthellae. Environmental stressors may destabilize the coral-zooxanthellae system resulting in the expulsion of zooxanthellae and/or loss of photosynthetic pigment within zooxanthellae, causing a condition known as bleaching. It is estimated that 1% of the worlds coral population is lost yearly, partly due to bleaching. Despite intensive research efforts, a single unified mechanism cannot explain this phenomenon. Although UVA and UVB cellular damage is well documented, UVC damage is rarely reported due to its almost complete absorption in the stratosphere. A small scale coral propagation system at the University of Maine was accidentally exposed to 15.5h of UVC radiation (253.7 nm) from a G15T8 germicidal lamp, resulting in a cumulative surface irradiance of 8.39 x 10(4) J m(-2). An experiment was designed to monitor the progression of UVC induced damage. Branch sections from affected scleractinian corals, Acropora yongei and Acropora formosa were submitted to histopathology to provide an historical record of tissue response. The death of gastrodermal cells and necrosis resulted in the release of intracellular zooxanthellae into the gastrovascular canals. Zooxanthellae were also injured as evidenced by pale coloration, increased vacuolization and loss of membrane integrity. The recovery of damaged coral tissue likely proceeds by re-epithelialization and zooxanthellae repopulation of gastrodermal cells by adjacent healthy tissue.

Biochemical Reference Intervals for the Resting State in the Adult Lobster Homarus americanus

ABSTRACT Clinical biochemistry is the measurement of body fluid constituents, and is considered an integral component of disease diagnosis in human and veterinary medicine. Biochemical reference intervals are the ranges of values of specific analytes at the population level that are expected to encompass 95% of “normal” individuals. However, how do we define a “normal” lobster? Homarus americanus (H. Milne Edwards, 1837) is a sublittoral ectotherm with a limited homeostatic ability to adapt to rapid environmental changes. An experiment was designed to measure the changes that may occur in selected analytes as wild lobsters “acclimate” to captivity. Biological variation was minimized by the maintenance of strict environmental conditions, uniform life stages, nutrition, and molt status. The results of this experiment suggest that elevations in hemolymph levels of glucose, L-lactate, crustacean hyperglycemic hormone, ammonia, ionic calcium, and a decrease in ionic potassium may be indicative of the stress associated with capture, handling, and emersion hypoxia. Although the data demonstrate a considerable intraspecific variation in most analytes during the acclimation process, these reference intervals may be useful in the interpretation of clinical findings in lobsters held in captivity for research or exhibition in public aquaria.

Determining the age of individual Lepeophtheirus salmonis (Krøyer, 1837) copepodids by measuring stored lipid volume; Proof of principle

Confocal microscopy has facilitated measurement of stained lipid volume in Lepeophtheirus salmonis copepodid larvae. Quantity of lipid, location and morphology of vesicles may allow an estimate of age and viability