Phenotypic Variation of the Zebrafish (Danio rerio, Actinopterygii: Cyprinidae) Skeleton in Response to Rearing Density

The response of individuals to novel or altered environments is defined as "phenotypic plasticity“. Plasticity is a fundamental character of the vertebrate skeleton (1). The Skeleton is active and dynamic throughout life and capable to adapt to mechanical forces and environmental changes. Here we study how rearing density affects skeletal development in zebrafish. From 30 to 90 days post fertilisation zebrafish were reared at three different densities. High density (HD) 32 fish/L, medium density (MD) 8 fish/L and low density (LD) 2 fish/L. A connected recirculating system ensured homogenous water chemistry. At the end of the experiment, animals were whole mount stained with Alizarin red S to visualise calcified tissues. Then, the entire skeleton was analysed for 113 malformations types (modified from (2)) and vertebral body malformations were subjected to histological analyses. The animals’ average standard length decreased with increasing rearing density. The HD group had the highest variety of malformations throughout the skeleton and the highest number of malformations per malformed specimens. The HD group particularly showed malformations in the caudal region of the vertebral column, such as dislocation of neural and haemal arches, missing distal fusion of arches and malformed neural and haemal spines. Histological analyses showed a variable size of muscle segments while arches and spines align with dislocated myosepta. Vertebral centra can extend over two myosepta, the opposite of diplospondyly. Interestingly, similar malformations have been described for tbx6 mutant (fssti1) and transgenic zebrafish in which the Notch pathway was inhibited (3). The mechanisms by which rearing density induces late vertebral column malformations similar to early, mutant-related, malformations remain to be elucidated.