GENETIC RESPONSE TO DIETARY MANIPULATIONS DURING TRANSFER TO

EXOGENOUS FEEDING IN ATLANTIC COD LARVAE

I. Rønnestad1, R. B. Edvardsen2, S. Applebaum1, S. Garg1, 3, C. Jolly1, Y. Kamisaka1, T. van der

Meeren4, and A.-E. O. Jordal1
1
Department of Biology, University of Bergen, N-5008, Bergen, Norway
2
Institute of Marine Research, Nordnes, N-5817 Bergen, Norway
3
Aqua Research Lab, Department of Zoology, University of Delhi, India
4
Institute of Marine Research, Austevoll, N-5392 Storebø, Norway

Introduction
The onset of exogenous feeding represents an ecologically, physiologically and developmentally
important transitional event for fish larvae. During this time, the digestive system is activated and
nutrient acquisition changes from endogenous yolk-sac to exogenous feed which is captured and
ingested. Larvae continue to undergo numerous developmental changes before they are fully
transferred into juveniles and these processes are well-known to be strongly impacted by diet. The
digestive system is responsible for digestion and absorption of ingested food and is exposed to all
dietary (+ other biotic) components which enter the gut lumen. There is scarce knowledge on the
changes in gene expression occurring in the digestive system in response to the onset of exogenous
feeding. Absorbed nutrients which enter the systemic circulation have profound effects on
developmental and cellular processes, but very little is known how dietary components affect
ontogenetic patterns of gene expression during critical stages of larval development. Therefore, our
aim of the present study was to examine the transcriptomic response of Atlantic cod, Gadus morhua,
larvae to the onset of exogenous feeding and to different diets.

Materials and methods

Newly hatched larvae were supplied from Parisvannet, Institute of Marine Research, transferred to
Bergen High Technology Center at 2 days post-hatch (dph), and divided in 4 stagnant tanks (water
volume of 160 l) with a density of 31 larvae.l-1. Larvae were reared at 8°C and under a 16:4h (light:
dark) photoperiod with 2h dusk and dawn. Feeding began at 2dph reffered as 0 day post feeding (dpf).
Prey densities were 5000 prey.l-1 and adjusted accordingly twice daily. In a linear regression design,
one tank remained unfed (A), one tank was fed wild-captured zooplankton, mainly copepod nauplii
(B), and one tank was fed zooplankton with algae from 1dph (C). Larvae from each tank were sampled
at -1, 0, 1, 2, 4, and 6dpf. Triplicate samples containing 125 pooled larvae were collected on dry ice
and flash frozen. Automated RNA isolation was used. RNA quality and quantity were determined
using the Agilent Bioanalyser 2100 and Nano Drop systems (A260/280 and A230/280). Total RNA
was subjected to separate direct cDNA synthesis protocols using Cy3 and Cy5 labelling and
hybridised to the 16K cod cDNA microarray before subjected to standard scanning procedures.
Microarray data analysis was performed using J-Express data analysis software (MolMine, Bergen,
Norway). Unpaired rank product analysis using 400 permutations on log2 values were performed to
identify gene candidates to be used for further analysis and validations.

Results and discussion

Onset of exogenous feeding resulted in transcriptomic responses in Atlantic cod larvae in all dietary
treatments. Correlation analysis clearly identified the influence of feeding regime at all specific time
points (0, 2 and 6dpf) (Fig. 1).
Fig. 1. Correlation analysis of Microarray Spot-pix data. Individual data are grouped according to
days post feeding (dpf).

Rank product analysis identified groups of genes which were differentially expressed (Table I), and
these results clearly indicated that more genes were differentially regulated in larvae fed zooplankton
only than those in larvae co-fed zooplankton and algae, whereas the number of differentially expressed
genes in the unfed larvae were lower.

Table I. Number of differentially expressed genes between feeding regimes indicated by rank product
analyses. (A) Unfed larvae, (B) zooplankton fed larvae, and (C) larvae fed zooplankton with
algae. Unfed larvae at 0dpf were used as the reference group (grey Italic letters).

Reference group Treatment group

Group ID dpf N Group ID dpf N No. of probe hits
A 0 9 A 2 3 969
A 0 9 A 6 3 1553
A 0 9 B 2 3 821
A 0 9 B 6 3 2028
A 0 9 C 2 3 803
A 0 9 C 6 3 1418
A 0 9 B and C 2 6 345
A 0 9 B and C 6 6 723
A 0 9 B and C 2, 6 12 N.a.

Conclusion
These results show that Atlantic cod larvae alter gene expression rapidly in response to the onset of
exogenous feeding and to the different dietary components.

Acknowledgements
This work was supported by Research Council of Norway grants #187281, 184602, 187063, and
grants from Helse Vest and the University of Bergen (NettMett). The publication benefits from
participation in LARVANET COST action FA0801.