ARRESTED DEVELOPMENT: The molecular and Endocrine Basis of Flatfish

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Flatfish species such as halibut, turbot and sole form a major focus of the diversification of European aquaculture industry. However, production has been severely hampered by biological problems in larval rearing. In the case of flatfish this is frequently associated with failed metamorphosis.The identification of reliable markers which can act as indices of flatfish metamorphosis progression implies that the markers identified should differentiate between normal and abnormal metamorphosis. Putative candidate markers have been identified and defined as molecular or morphological indicators which changed between the pre-metamorphic larvae (stage 8) and post-metamorphic juveniles (stage 10). Useful markers for metamorphosis should be those which differentiate life-stages and are diagnostic of normal and abnormal metamorphosis. Numerous markers have been identified during ARRDE which change during metamorphosis in Atlantic halibut: Musculo-skeletal system - Markers for metamophosis:- YES (MLC1, 2 and 3, fTnT); Invasive:- YES (larvae have to be killed), Candidate indicators of normal/abnormal metamophosis:- YES (efTnT but further validation required). Skeletal system - Markers for metamophosis:- YES; Invasive:- NO (live animal can be assessed); Candidate indicators of normal/abnormal metamophosis:- YES (Abnormal stage 8 fish abnormal remodelling of the frontals shows bilaterally equal activity of the osteoclasts. In abnormal stage 9 fish the sinistral eye has not migrated, the frontals are symmetrical and there is equal lateral osteoclastic activity in the frontal processes). Gastrointestinal system - Markers for metamophosis:- YES (pepsinogen and trypsin); Invasive:- YES (larvae have to be killed); Candidate indicators of normal/abnormal metamophosis:- YES (further validation of the use of pepsin and trypsin required, but they are significantly different in normal and abnormal stg, 8, 9 and 10 halibut). Skin - Markers for metamophosis:- YES (epidermal keratin); Invasive: YES (larvae have to be killed); Candidate indicators of normal/abnormal metamophosis: Unlikely to be useful marker of normal/abnormal metamorphosis. Blood - Markers for metamophosis:- YES; Invasive:- YES (larvae have to be killed); Candidate indicators of normal/abnormal metamophosis:- No information is available from abnormal larvae. It is expected that markers will be aplicable to flatfish in general but requires further testing. Markers which are diagnostic of abnormal metamorphosis of flatfish are difficult to encounter and substantial validation of their potential use is still required. The generation of a tool to improve, quality, health and predictability of fish larval production have important implications on improving sustainability of aquaculture which is frequently located in rural areas with limited employment opportunities. Aquaculture represents a sustainable and probably the only means of generating high quality fish available to all Europeans with its recognised beneficial impact on health.

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.

A microarray was constructed using: (i) Approximately 1152 clones from subtractive libraries enriched in sequences up or down-regulated at metamorphosis and (ii) 480 clones randomly picked from a non-normalised cDNA library prepared from metamorphsosing larvae. High quality sequence data has been obtained for 970 of the arrayed clones. This shows some redundancy siuch that the entire array is estimated to represent 500-600 genes. Database seaching using the sequences generated has allowed identification of 296 of the arrayed genes. Preliminary screening of the microarray has been successful indicating that it is suitable for high throughput analysis of gene expression. Thus it might be used to analyse how changes in diet or other aspects of culture affects the expression of a large panel of genes.

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