Antagonistic activity of bacterial isolates from intestinal microbiota of Atlantic cod, Gadus morhua, and an investigation of their immunomodulatory capabilities

In an effort to identify potential probionts, four bacterial strains obtained from the intestinal tract of wild-caught Atlantic cod, Gadus morhua , were tested for their antagonistic activity against Vibrio anguillarum and Aeromonas salmonicida , two of the most common bacterial pathogens in cod aquaculture, using a well-diffusion agar assay at two incubation temperatures: 13 and 20 °C. The tested bacteria exhibited dissimilarity in their inhibitory action against the target pathogens – an enhanced activity was particularly observed for strains GP11 and GS11 at the highest incubation temperature. Based on the 16S ribosomal DNA gene sequence analysis, the strains showed high similarity to Psychrobacter sp. (strain GP11), Shewanella sp. (GS11), Photobacterium sp. (GP31) and Vibrio sp. (GV11). The incubation of Atlantic cod head kidney cells with the heat-inactivated probiotic strains resulted in the differential expression of immune-response genes that are related to bacterial defence and inflammation. Thus, the gut-derived bacterial strains have probiotic potential and their possible immunomodulatory capabilities could be determined under in vitro conditions.     

Experimental mycobacteriosis in Atlantic cod, Gadus morhua L

Piscine mycobacteriosis causes losses in a number of fish species both in the wild and in aquaculture worldwide. Mycobacterium salmoniphilum infections have on several occasions been reported in farmed Atlantic salmon, Salmo salar L. The present study tested and confirmed the susceptibility of Atlantic cod, Gadus morhua L., an important yet relatively novel aquaculture species, to infection with M. salmoniphilum. Atlantic cod injected intraperitoneally with a suspension of this bacterium were maintained together with cohabitant (COH) fish in a flow-through marine water system at 10-11 °C. The fish were supervised daily and samples taken at 2, 7, 14, 23, 34 and 53 weeks post-infection and examined pathologically, bacteriologically and using molecular biology. Injected mycobacteria were re-isolated in high concentrations from both injected and COH fish groups. Death attributable to mycobacterial infection was observed in both injected (47%) and COH (28%) fish groups. Extensive development of granuloma in visceral organs, mainly the mesenteries, spleen, kidney and liver (lesser extent) and at later stages of the infection in heart tissues and gills, was observed in both injected and COH fish. Granulomas underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Acid-fast bacilli were detected in both granulomas and non-granulomatous tissues. This study confirms that Atlantic cod is highly susceptible to M. salmoniphilum infection and that this bacterial species may be a threat to cod both in the wild and in the aquaculture.     

Activation of intestinal epithelial cells in Atlantic cod,Gadus morhua,induced by algal derivatives

This study investigated the influences of two algal derivatives, alginic acid and fucoidan (FUC) on the activities of the intestinal epithelial cells (IEC) of Atlantic cod. The cells were isolated from three regions of the intestines, and primary cultures were incubated with the derivatives followed by temporal analysis of cellular activities. Epithelial cell survival in the fore and hind intestine was not significantly affected by the algal derivatives at both time points (3 and 24 h postincubation, hpi). Cellular survival in the mid‐intestine decreased significantly at 3 hpi. Stimulation of myeloperoxidase (MPO) activity of IEC induced by the algal derivatives was observed to be region dependent. Myeloperoxidase activity in the cells from fore‐ and mid‐intestines was not significantly affected by the two algal derivatives. Both algal derivatives significantly stimulated the activity at 3 hpi in the hind intestinal cells, and FUC‐exposed cells remained significantly elevated until 24 hpi. The results showed that algal derivatives could be applied as potential immunostimulants in cod with stimulatory action of MPO activity in the mucosal immune cells of the hind intestine.     

The effect of dietary chitin on the autochthonous gut bacteria of Atlantic cod ( Gadus morhua L.)

In the present study the impact on autochthonous (adherent) bacteria in proximal intestine (PI) and distal intestine (DI) of Atlantic cod ( Gadus morhua L.) was evaluated following feeding of a control diet and a diet supplemented with 5% chitin. The autochthonous gut bacteria were investigated using denaturing gradient gel electrophoresis (DGGE). Analysis of the microbiota associated with PI and DI of Atlantic cod indicate that dietary chitin modulate the intestinal bacterial community. For example, band 25 ( Escherichia coli–like), band 14 ( Anaerorhabdus furcosa–like) and band 29 (uncultured bacterium–like) in PI were depressed by dietary chitin (P < 0.05). The number of bands (23.7 ± 5.4) in DI of fish fed chitin was marginally higher than the control fish (16.7 ± 2.1) (P = 0.065), and the relative abundances of band 6 (swine faecal bacterium–like) were marginally stimulated by dietary chitin (P = 0.095). Furthermore, the present study reports several novel sequences not previously reported in the gastrointestinal tract of Atlantic cod. Whether the dietary effect of chitin on gut bacterial community has any positive effect of fish health merits further investigations.     

Carnobacterium maltaromaticum vs. Vibrio (Listonella) anguillarum in the midgut of Atlantic cod (Gadus morhua L.): an ex vivo study

The present investigation addresses whether the midgut (MG) of Atlantic cod (Gadus morhua L.) is an infection route for Vibrio (Listonella) anguillarum serotype 02 β and if Carnobacterium maltaromaticum, a probiotic bacterium, can out‐compete the pathogen and modulate the autochthonous MG microbiota. This was investigated by using an ex vivo method the intestinal sac, utilized previously in studies on Atlantic cod and Atlantic salmon (Salmo salar L.). Exposure of the MG to V. (L.) anguillarum did not reveal any cell damage indicating that the MG does not appear to be an infection route for V. (L.) anguillarum in healthy Atlantic cod. This finding together with previous observations on Atlantic cod and Atlantic salmon indicate that intestine as an infection route might vary between these two species. When the MG was exposed to C. maltaromaticum, no cell damage or cellular disruptions were observed. As budding from the apices of microvilli was observed in all treatments exposed to bacteria, we suggest that budding might be involved in the primary barrier against bacterial infection. However, to clarify this hypothesis, further studies are needed. Exposure of the MG to the probiotics and pathogenic bacteria indicated that C. maltaromaticum, to some extent, is able to out‐compete V. (L.) anguillarum but the topic merits further investigation. Analysis of the MG microbiota after sterile saline solution and bacterial exposure indicates that bacteria related to Staphylococcus sciuri belong to the autochthonous gut microbiota in Atlantic cod.     

Environmental influences on melatonin secretion in Atlantic cod (Gadus morhua L.) and their relevance to commercial culture

This study investigated the effects of photoperiod and temperature on plasma melatonin secretion in Atlantic cod (Gadus morhua L.). Initial work confirmed the presence of a diel profile of melatonin synthesis, with elevated levels during the dark phase. Unusually for fish, the peak in plasma melatonin occurred towards the end of the dark phase, which is indicative of a type `A' melatonin profile. When exposed to 60 hours of continuous darkness a clear endogenous rhythm of melatonin synthesis was observed, which continued for 4 cycles with a periodicity which, approximated to 24 h. When acclimated to varying temperatures (4, 8, 12 or 16 °C) no variation in melatonin production was seen, however, body size appeared to be an important influence, with the smallest fish exhibiting significantly higher levels of dark phase melatonin. Finally, the application of additional night-time illumination to cod maintained in sea cages i.e. without blackout, did not significantly reduce dark phase plasma melatonin, suggesting that cod are less sensitive to photoperiod manipulation in cages than salmonids.     

Growth and gut morphology of diploid and triploid juvenile Atlantic cod (Gadus morhua)

The objective of this paper was to compare the growth and gut morphology of juvenile diploid and triploid Atlantic cod (Gadus morhua) reared under similar conditions. Individually tagged 36‐week‐old diploid (mean weight 49.3 ± 13.8 g) and triploid (mean weight 43.6 ± 11.2) juvenile cod were measured at intervals during a 29‐week growth trial. Data for weight, length, condition factor (K), hepato‐somatic index (HSI), gonado‐somatic index (GSI), Relative Gut Length (RGL) and pyloric caeca number were collected and results were analysed in relation to ploidy status, gender and family contribution. At the end of the experiment, only one family (M2xF3) had many representatives with a relatively even distribution of sexes and ploidies. Diploid females were significantly heavier and had higher K than triploid females in the M2xF3 family (body weight 371.2 ± 120.2 vs. 298.4 ± 100.7 g; K 1.1 ± 0.1 vs. 0.93 ± 0.1), but no differences were found between diploid and triploid males. In the other families (pooled data), no differences in body weight were found between the ploidy groups. In general, triploids had a shorter intestine (RGL) and fewer pyloric caeca than their diploid siblings regardless of gender suggesting possible impairments in nutrient utilization and growth.     

Dietary plant protein utilization in Atlantic cod, Gadus morhua L.

The present trials aimed to investigate the effects of replacing fish meal with plant proteins in diets for cod, using a regression design where fish meal constituted the control. The plant protein diets were formulated to meet the amino acid requirements according to NRC (1993) and contained corn gluten meal, soybean meal, a mixture of these, or a mixture of wheat gluten meal and soybean concentrate. The plant protein fraction constituted up to 440 g kg⁻¹ of the extruded diet. Two feeding experiments were conducted, one at high (11 °C) and one at low (6.5 °C) temperature. High growth and feed utilization were obtained in all diet groups at both temperatures. However, only in Exp.1 (11 °C), growth and feed utilization were linearly reduced by increases in dietary soybean and corn gluten meal, while no such effect was detected in Exp.2 (6.5 °C). Reductions in protein retention were seen at both temperatures with use of all evaluated plant protein sources. Due to large amounts of plant protein ingredients in diet, differences in diet amino acid composition were seen. These were partly reflected in the muscle free amino acid pool. Dietary plant ingredients did not affect whole body, liver or muscle proximate compositions, or liver indices. In both experiments, blood parameters were within the range of earlier reported normal values and indicated, together with low mortality, good fish health status. The results show that there is high potential to use protein‐rich plant ingredients in diets for Atlantic cod.     

Heritability of economically important traits in the Atlantic cod Gadus morhua L

During the development of breeding programme for Atlantic cod Gadus morhua L., in Iceland, genetic parameters were estimated for 1402 individuals, which were assigned with DNA profiling to 140 dams and 70 sires. The cod was reared in cages on the eastern and western coasts of Iceland from 2004 to 2005. At the average body weight of 1.8 kg, the estimated heritability (h² ± SE) for body weight, gutted weight and the condition factor (CF) were 0.31 ± 0.06, 0.34 ± 0.04 and 0.24 ± 0.06 respectively. Genetic correlation (r_G) in body weight between the two rearing locations was estimated as 0.95, which reflects a low G × E interaction. The estimated heritability for hepatosomatic index (HSI) and fillet yields was 0.061 ± 0.04 and 0.04 ± 0.04 respectively. The HSI and fillet yields were highly genetically correlated with body weight or 0.67 and 0.82 respectively. The genetic correlation between the CF and body weight was estimated as 0.31. There appears to be substantial amount of additive genetic variation for body weight suggesting that selection is likely to be successful. Low heritability for fillet yields and the HSI indicates less promise of genetic improvement. Assigning of parentage to individuals with DNA profiling was 80% successful.     

Digestive capacity and compensatory growth in Atlantic cod (Gadus morhua)

The objective of this study was to examine whether digestive capacity correlates with growth rate in Atlantic cod (Gadus morhua). Ninety fish (1035±240 g; 478±36 mm) were assigned to one of three treatments: deprived of food for 5 weeks and then re-fed for 24 days, deprived for 10 weeks and then re-fed for 24 days, or fed for 24 days without deprivation (controls). Three times a week during feeding, the fish were provided with meals of capelin (Mallotus villosus) in excess. Within each treatment, ten fish were sacrificed before and twenty after the feeding period. The relative masses of the pyloric caeca, intestine, and white muscle were determined. Cytochrome c oxidase (CCO), citrate synthase (CS), and nucleoside diphosphate kinase (NDPK) activities were assayed in the pyloric caeca, intestine, and muscle as was trypsin activity in the pyloric caeca. During the re-feeding period, fish that had been deprived of food for 10 weeks showed compensatory growth, growing 1.9 times faster than the controls (0.94±0.26 versus 0.50±0.31% body mass ⋅ day⁻¹). Fish that displayed compensatory growth had a lower relative white muscle mass than controls (66.5±4.1% vs. 69.6±7.4%) while the relative masses of the pyloric caeca (2.21±0.49% vs. 1.78±0.31%) and intestine (0.95±0.20% vs. 0.77±0.15%) were greater than the controls, suggesting that the sizes of these digestive tissues might correlate with compensatory growth capacity. Citrate synthase in the pyloric caeca was the only enzyme that showed higher activity during compensatory growth (8.57±0.94 U ⋅ g tissue⁻¹ compared to 7.13±1.03 U ⋅ g tissue⁻¹ in the control group). This suggests that aerobic catabolic capacity of pyloric caeca could be related to growth capacity during recovery, possibly via the energetic cost of digestive enzyme synthesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stress response to anthropogenic noise in Atlantic cod Gadus morhua L.

The potential effects of anthropogenic noise on the physiology of Atlantic cod have not been well described. The aim of the present study was to investigate the impact of anthropogenic noise on Atlantic cod stress response using cortisol as a biomarker as well as on broodstock spawning performance. Results showed that artificial noise consisting of a linear sweep from 100 to 1000 Hz can induce a transient and mild cortisol elevation with a clear noise intensity dose response. In all cases plasma levels returned to baseline levels <1 h post sound exposure. Daily exposure to a similar intensity and frequency noise range applied habitually to a broodstock population during the spawning window resulted in a significant reduction in total egg production and fertilisation rates thus reducing the total production of viable embryos by over 50%. In addition, a significant negative correlation between egg cortisol content and fertilisation rate was observed. These results confirm that cod can perceive noise generated within a frequency range of 100–1000 Hz and display a heightened cortisol plasma level. In addition, anthropogenic noise can have negative impacts on cod spawning performances.     

Mercury comparisons between farmed and wild Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Wild and farmed Atlantic salmon ( Salmo salar L.) and Atlantic cod ( Gadus morhua L.) were collected to assess changes in mercury with size in wild vs. farmed fish. Mercury concentrations were compared with Health Canada and United States Environmental Protection Agency consumption guidelines. Lipid dilution of mercury was examined by comparing lipid-extracted (LE) and non-lipid-extracted (NLE) flesh samples in both farmed and wild fish. Mercury concentrations in the flesh and liver of farmed salmon were significantly lower than concentrations in wild salmon of similar fork length ( P <0.001), possibly due to growth dilution in rapidly growing farmed fish. Mercury concentrations were higher in LE tissue compared with NLE ( P <0.05), suggesting lipid dilution of mercury in farmed fish with a high lipid content. Farmed cod, which do not grow more rapidly than wild cod, did not have significantly different flesh and liver concentrations compared with wild cod of similar fork length ( P >0.05). Between species of farmed fish, cod had significantly higher mercury concentrations than salmon ( P <0.05), but neither farmed nor wild salmon mercury concentrations exceeded federal consumption guidelines. These results suggest that rapid growth rates and a high lipid content may play important roles in regulating concentrations of contaminants such as mercury.     

Effect of Antarctic krillmeal on quality of farmed Atlantic cod (Gadus morhua L.)

Farmed Atlantic cod with a mean weight of 4.8 kg were maintained for 9 weeks in sea cages and fed diets where the dietary fishmeal component was substituted with increasing proportions (0%, 22%, 63% and 100%) of meal from Antarctic krill, Euphausia superba. Wild‐caught cod were included in the study as external control. At termination of feeding, all fish were slaughtered and muscle pH was immediately recorded. The fish were then stored on ice for three days and assessed for muscle pH and objective (skin and fillet colour, and fillet texture) and subjective (sensory evaluation) quality criteria. Replacement of fishmeal with krillmeal in the diets resulted in the skin colour above and below the lateral line to be more red than the control group without krillmeal substitutions, even though this difference was not significant, and with more yellow hue. Additions of krillmeal increased the muscle whiteness and yellow hue compared with the control group and wild fish. There was no difference in red hue between the groups. Muscle pH, texture or sensory attributes were unaffected by dietary inclusion level of krillmeal. Wild‐caught cod deviated in several aspects from the farmed cod. It is concluded that the replacement of fishmeal with Antarctic krillmeal in the diets two months before slaughter did not move the sensory attributes more towards wild fish.     

Inter‐individual variation in the dominant intestinal microbiota of reared Atlantic cod (Gadus morhua L.) larvae

The diversity of the microbiota in 15 Atlantic cod (Gadus morhua L.) larvae from three different hatcheries with variable food type and water treatment was characterized with culture dependent and independent approaches. The culture‐dependent approach was phenotypic characterization of bacterial isolates based on 16 tests, and the culture‐independent approach was terminal restriction fragment length polymorphism (T‐RFLP) of PCR amplified 16S rRNA. Both approaches revealed large inter‐individual differences in the microbiota of cod larvae. Data from real time PCR indicate substantially lower variation in bacterial number larva^?1 both within and between hatcheries than the data on colony forming units, resulting in an estimated four orders of magnitude variation in culturability between larvae. Considerably higher richness and diversity were observed with T‐RFLP than with phenotypic characterization, and no correlation between the two approaches was found for diversity indexes calculated at the individual level. Dissimilarity in the composition of the microbiota between individuals from the same rearing environment was comparable to the dissimilarities between hatcheries. The large inter‐individual variation is important to consider when designing experiments, as pooling of individuals will completely mask this variability. We conclude that colonization of individual cod larvae by bacteria seems to be a stochastic and not a deterministic process, which is not well understood.     

Occurrence of Francisella piscicida in farmed and wild Atlantic cod, Gadus morhua L., in Norway

Francisellosis, caused by the bacterium Francisella piscicida , has become one of the most serious diseases in Atlantic cod production in Norway. The major aim of this study was to determine the distribution of F. piscicida in farmed and wild fish in areas with cod farming along the Norwegian coast, and its occurrence in cod from areas without cod farming. Two real-time PCR assays, targeting the 16S rRNA gene and the FopA gene of F. piscicida , were developed since sensitive and specific diagnostic tools are required for detecting asymptomatic carriers of the bacterium. A total of 422 wild cod from 13 sampling areas and 955 farmed cod from 10 areas along the coast of Norway were examined. Using the real-time polymerase chain reaction (PCR) assays, F. piscicida was detected in wild populations of cod from all counties examined south of Sogn og Fjordane in southern Norway (overall prevalence 13%, n  = 221). Wild cod north of Sogn og Fjordane were negative for the bacterium ( n  = 201). Farmed cod from most parts of Norway were F. piscicida positive. The apparent absence of the bacterium in wild populations of cod in the northern parts of Norway and its widespread occurrence in wild cod from southern parts of Norway is believed to relate to differences in seawater temperatures.     

Muscle growth of triploid Atlantic cod juveniles (Gadus morhua)

This study has investigated the muscle growth of diploid and triploid Atlantic cod (Gadus morhua) juveniles raised in replicate tanks over a period of 29 weeks and analysed at three sampling points (February, June and September). Data for weight, length, condition factor (K), muscle fibre growth and myogenic progenitor cells (MPCs) number were collected and results were analysed in relation to body growth and ploidy status. Diploids were significantly heavier than triploids throughout the trial (~10–20%) and had K in June and September samplings. Over the whole period, the rate of muscle fibres' recruitment was 318 fibres per day and 252 fibres per day for diploid and triploid cod respectively. The larger body weight of diploids resulted in a total number of fast fibre number of 114 979 compared to 91 086 in triploids. The average diameter of the 2.5% of the smallest fibres (2.5th percentile) was higher in diploids than triploids at the start of the trial, with a reversed picture for the average of the upper 2.5% (97.5th percentile) at the end of the trial. The probability density function of the estimated muscle fibre diameters showed similar fibre size distribution between size‐matched diploids and triploids at all sample points. The peak fibre diameter was approximately 25 μm in February and increased to approximately 50 μm in June and September, irrespectively of ploidy. Pax 7 were used as molecular markers for MPCs. A positive correlation between Pax 7⁺ cells and total body length was observed only among triploid fish at the onset of the experiment.     

Spawning‐related mortality in captive Atlantic cod (Gadus morhua L.)

Two year‐classes of Atlantic cod were reared in indoor tanks from November 2002 to November 2004. The average annual mortality was 21% and 36% for males and females respectively. The highest monthly mortality rates in females were observed around and during the spawning periods from March to July, maximally 7% and 20% for year‐class 2001 and 18% and 34% for year‐class 2000. The mortality in males was less dependent on season, reaching a maximum of 7% and 5% month^?1 for year‐class 2001 and 2000 respectively. The maximum mortality during spawning increased with age from: 7%, 19% and 34% per month for 2‐, 3‐ and 4‐year‐old females respectively. The average gonadosomatic indices in 3‐ and 4‐year‐old mature females that died during the spawning periods were 22% and 26% respectively and most of them were spawning when they died. The results suggest that egg‐retention may have been the main cause of death in females during spawning.