THE CHANGES IN COD INTESTINAL MICROBIOTA UPON CAPTURE

This study documents the intestinal changes in the gut of wild Atlantic Cod, Gaduas morhua, upon capture and artificial feeding. Over the years, this species has become increasingly important for mariculture in the northern hemisphere. Presently, wild Cod are caught and brought to sea-pens were they are fed an artificial diet to increase weight and size before slaughter. This practice also supplies a year-round Cod source. Past studies indicate that marine fish caught from the wild and fed artificial feed may change the intestinal microbiota in the caught stock. It is well-established that the microbiota of fish intestines plays an important role in the growth, survival, health and nutrition of a host, supplying nutrients, fatty acids, extracellular enzymes and vitamins. The intestinal communities of wild-caught and penned Scottish/Norweigian Salmon has been documented but here the changing intestinal communities of wild Atlantic Cod acclimatizing to artificial rearing will be assessed for the first time.

70 cod were caught off the coast of Norway and brought to a large indoor tank supplied with 250m seawater. Initial intestinal samples were collected from 8 fish before the remaining were distributed into 6 different tanks. They were left to acclimatize for 1 week before two different feeding trials began for 5 weeks. 3 tanks were fed with a formulated powder mixed with sausage and herring, these were fed to satiation, and 3 tanks were starved for 5 weeks. 8 fish were sampled from each feeding trail. After being euthanized, the intestinal tract was removed using aseptic procedure. The fore, mid and posterior sections were isolated. These were washed in a sterile o.o1 M phosphate-buffered saline solution and the contents was removed. These were kept on ice until analysis. The empty intestine was then flushed dissected into small pieces. This remained sterile for molecular analysis.

In order to collected quantitative data, the intestine contents were subject to a ten-fold dilution and plated onto Difco marine agar. The colonize that formed were observed for 3 weeks and counted. In addition a DAPI flurochrome stain was applied to fixed samples and photographed under a microscope.

Qualitative data was collected using DGGE (Denaturing gradient gel electronphoresis) and PCR-amplified genes coding for 16S rRNA. These would allow analysis of any intestinal community changes. DNA was isolated from the intestinal wall and content, and a primer amplified the 550-bp fragment in the rDNA. An annealing temperature gradient PCR was performed to find optimum temperature of amplification to be 55.8°C. The products of the PCR were used to produce a DGGE. There were four different analysis conducted; the posterior content, posterior wall, mid content and mid wall samples. The gels were stained with a diluted SYBR green stain. These were also photographed. Products were also purified using a PCR centrifuged filter and subjected to BLAST.

It was recorded that there was an increase both types of counts conducted when comparing the mid to the posterior intestine. This was recorded in all three feeding groups. Only the mid content compared to the mid wall showed a decrease in abundance and this only occurred in the starved trial. The band numbers in the DGGE were less in the fed group than the initial-capture group. The two groups showed similarity values of less than 50% however. The starved group however had almost the same number of bands as the initial group; this suggested that starvation had not effected the microbial structure.

From the quantitative studies, it was seen that there was no difference between the number of directly counted groups when comparing the initial-caught group with the fed and starved group. It was presumed that the resistance to change was caused by stability within the intestinal environment, as has been shown in previous studies. It was also discussed that the starved Cod were still found to contain hard digested matter, suggesting that a longer period of starvation than the proposed 42 day period should be implemented in later studies. This study reveals that the microbial community of Atlantic Cod remains at a relatively constant level despite dietary changes. The paper further suggests that the persistent microbiota might be ‘sentinal organisms’.

The qualitative data determined that there was a difference in the number of bands between the initial-capture group and the fed group. This was unexpected as the number of counts showed no significant difference in the quantitative data set. It was proposed that the abundance of microbiota  remained the same but the community shifted. This is consistent with previous studies conducted upon various freshwater and marine fish.

Finally the paper notes that the number of culturable microbes were very low when compared to the number of microbes identified by the DGGE. This was shown to be down to the number of anaerobic bacteria present in the intestine.

This paper is intriguing as it uses an array of techniques to consider the microbial composition of wild cod intestines in association with a change in diet.

Anushu et al. 2011. Changes in the Intestinal Microbiota of Wild Atlatnic cod Gadus morhua L. upon captive rearing, Microb. Ecol. 61: 20-30