The effect of fixation on the morphology of the late premolt and early postmolt cuticle of the blue crab, Callinectes sapidus

UNCW Author/Contributor (non-UNCW co-authors, if there are any, appear on document)
Shannon Modla (Creator)
The University of North Carolina Wilmington (UNCW )
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Richard Dillaman

Abstract: The dorsal carapace from late premolt (D3) and early postmolt (1 hr) blue crabs (Callinectes sapidus) was used to study the effects of fixation on the morphology of the epicuticle, exocuticle, and hypodermis. Tissues were freshly dissected, quick-frozen in liquid nitrogen, or lyophilized. They were then treated with a standard fixation consisting of sequential fixation in glutaraldehyde, osmium tetroxide, tannic acid and uranyl acetate or with a uranyl acetate primary fixative. Lyophilized-unfixed and lyophilized-rehydrated tissues were also examined. Treatments were viewed using transmission and scanning electron microscopy. Standard fixation preserved both cuticle fibers and cellular elements. Results varied due to the impervious nature of cuticle and the formation of a permeability barrier by 1 hr postmolt. Uranyl acetate fixation greatly improved the contrast of fibers and enhanced the visualization of calcification initiation sites at the epicuticle-exocuticle interface and along the interprismatic septa (IPS), but it did not preserve the hypodermis. The IPS varied in staining intensity among samples, but overall fixation was more uniform than standard fixation. Both treatments revealed a diverse collection of fiber types. Anaglyphs showed that horizontal fibers, oriented parallel to the cuticle surface, rotated in successive planes while vertical fibers were oriented perpendicular to the cuticle surface. Of the vertical fiber types observed, some were associated with pore canals, others were not, and certain ones were restricted to specialized regions of the cuticle. Quick-freezing caused voids to occur between horizontal fibers. Voids were accentuated by lyophilization. The cuticle was more distorted by voids when it was quick-frozen or lyophilized and treated with standard fixation than with uranyl acetate fixation. Unfixed lyophilized samples had a unique tubular morphology that exhibited a lamellar periodicity. Rehydration of lyophilized cuticles reconstituted the fibrous matrix, with all fiber types being preserved except the IPS. Fibers in this treatment appeared most similar to uranyl acetate fixations but were more dispersed. Although the tubular morphology of unfixed, lyophilized samples appears to be an artifact of quick-freezing, it speaks to the highly hydrated nature of the cuticle at the time of ecdysis.

Additional Information

A Thesis Submitted to the University of North Carolina at Wilmington in Partial Fulfillment of the Requirement for the Degree of Masters of Science
Language: English
Date: 2009
Blue crab, Blue crab--Morphology, Callinectes--Morphology, Ecdysis, Molting
Blue crab
Blue crab -- Morphology
Callinectes -- Morphology

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