Date of Award

1999

Document Type

Thesis

Publisher

Edith Cowan University

Degree Name

Bachelor of Science Honours

Faculty

Faculty of Communications, Health and Science

First Supervisor

Dr Annette Patak

Abstract

Yabbies have the ability to regenerate lost limbs. Loss of a limb results in the growth of a limb bud. Development of this new limb involves many developing stages until the new limb becomes morphologically and physiologically virtually indistinguishable from its predecessor. In the yabby, the claw becomes functional after the first moult when the dactyl becomes moveable. However, the muscle fibres are able to develop force and appear to be functioning before the dactyl becomes moveable and the claw is operational (West et al., 1995). This study aimed to document morphological stages of claw regeneration and determine sequence and protein appearance of all fibre types in the claw closer muscle of yabby, Cherax albidus. The regenerating limbs were harvested at strategic stages, and the contralateral pristine claw was taken as a control. The stages of re-growth sampled were an unsegmented limb (papilla), a limb with a closed dactyl, an early stage of the limb with an open dactyl, and a fully regenerated limb. Fast (SL< 4µm) and slow (SL> 8µm) fibres were isolated from pristine and regenerating claw muscle of the yabby, C. albidus. Muscle tissue was taken from the papilla and closed dactyls. Myofibrillar proteins in fibres from the pristine claw closer muscle were identified using SDS-PAGE gel electrophoresis. Fibres contained numerous isoforms of structural and regulatory proteins in assemblages correlated with fibre type. One fast (F) and one slow (S2) fibre types were identified. All F fibres possessed two isoforms of paramyosin (P1 and P2), while all slow (S2) fibres contained only the P2 variant. Slow (S2) fibres were distinguished by the distribution of a large isoform of troponin T (T1), while fast fibres contained an unidentified 75K protein. Fast and slow fibres also differed in the assemblage of other regulatory proteins. The ratio between myosin heavy chain to actin in both fast and slow (S2) fibres was greater than one. During the initial stages of claw regeneration, the tissue contained undifferentiated muscle mass surrounded by large numbers of dense structures that were possibly haemocytes or nuclei. Protein profiles by SDS-PAGE revealed an extremely large amount of an 88K protein. The closer muscle does not differentiate until the closed dactyl stage where myofibrils were found to contain scattered fragments of sarcomeres. Sarcomere length of these myofibrils were of intermediate type. Mature, fully differentiated myofibrils (complete sarcomeres) had a short sarcomere length characteristic of fast fibres. Myofibrillar protein analysis by SDS-PAGE could not confirm fibre type. Most proteins from both fibre types were present at this stage. Throughout this phase, the ratio of myosin heavy chain to actin was less than one. Once the dactyl opened, the appearance, sarcomere lengths and protein profiles of the muscle fibres were indistinguishable from pristine fibres, and both fast and slow fibres were present. However the ratio of myosin heavy chain to actin in slow muscle fibres was approximately equal to one, fast muscle fibres contained a ratio greater than one. Results obtained also showed there was a greater proportion of slow fibres (65%) in the open dactyl than in pristine claws (10%). The muscle fibres of fully regenerated claws were indistinguishable in all aspects from pristine claws.

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