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Staging scheme for larval Atlantic halibut

It was a major challenge but an obvious necessity that we be able to apply a meaningful standardization to the immense numbers of halibut larvae to be sampled and analyzed. Fish larvae display individual rates of development, and these differences increase with time. Any kind of biologically meaningful sampling must take this into account.

Furthermore a description of what was normal vs what was abnormal at each developmental stage would precede an understanding of which markers could be used to identify the normal progression of metamorphosis in flatfish.

The objectives of this work were to standardize sampling procedures between labs, and to standardize reporting. Descriptions of normally and abnormally metamorphosing larvae identified potentially useful markers which could characterize the progression and success of metamorphosis.

To establish developmental stages independent of eye migration, 180 sibling halibut larvae, spanning from first feeding til settlement, were examined, cleared and stained for ossification and the cranial development was recorded. In particular the development and fusion of the cranial bones were followed, independent of eye migration in normal and abnormally metamorphosed halibut larvae. Morphometrics were correlated with internal cranial development and then validated on a further two groups of halibut larvae (n=23, n= 101) (Sæle et al. 2004).

Stages 5-9 from first feeding to settlement were defined (Sæle et al. 2004), comprising premetamorphosis to climax metamorphosis, with significant morphometric differences between stages and based on the appearance of ossified elements. These were then correlated with age, size and especially myotome height for easy application in other experiments. Morphological development and cranial ossification generally coincided. The order of ossification of cranial structures was: jaw structures, hyoid arch, opercular bones and structures of the neurocranium. The Frontale exhibited torsion correlated with eye migration, but calcification began earlier and full calcification was independent of ocular displacement.

There was a linear relationship between stage and myotome height (R2 = 0.86) and stage and standard length (R2 = 0.80). The stage definitions were validated on two groups (n = 23, n = 101) of commercially produced larvae. Because metamorphosis is protracted in halibut, use of these robustly defined stages and especially myotome height should help standardize sampling and analysis between experiments and between producers.

The trajectory of juvenile development appears fixed by Stage 8. Abnormal, arrested or delayed metamorphosis is obviously manifested as variations on the themes of misplacement of the anterior dorsal fin, incomplete eye migration, malformation of the cranium and malpigmentation. Normally, the anterior dorsal fin will be continuous with the head below (on the abocular side of) the migrated eye and both eyes are on the ocular side.

To establish the order of events involved in the most prominent feature of flatfish metamorphosis, eye migration, we examined 34 normal and 4 abnormal fish ranging from the least to the most developed stages for which normality could be ascertained visually (Sæle et al. 2006). Serial sections were made of the head of each specimen and eight sections representing the same eight regions were digitized and transformed to permit the use of morphological landmarks.

To examine the postembryonic remodeling of neurocranial elements, 24 fish larvae comprising 9 abnormal and 15 normal halibut, were analyzed for osteoclastic activity by using Tartrate-resistant acid phosphatase (TRAP). Serial transverse sections were stained for TRAP activity and the area measured by stereology (Sæle at al 2006). Cell proliferation in the neurocranium was visualized by immunocytochemistry using anti-PCNA (anti- proliferating cell nuclear antigen) on sections adjacent to those used for osteoclastic activity.

There are regional and lateral differences in the osteoclastic activity which is remodelling the frontals to accommodate the migrating eye in normal versus abnormal fish in Stages 8, 9 and juvenile halibut. In arrested halibut lacking eye migration, TRAP expression is even and constant between the regions both in Stage 9 and in the juvenile. There is an apparently large increase in activity from Stage 9 to the juvenile, but the TRAP expression in the arrested juveniles is highly variable.

Our findings indicate a tissue to tissue communication from the eye to osteoclasts control the osteoclast activity. These elements may start acting by Stage 7 or earlier, and have measurable action even as the final phenotype is being attained.

Related information

Reported by

University of Bergen
Dept Fish Mar Biol
5020 Bergen
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