Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

Abstract

SHORT ABSTRACT The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species. LONG ABSTRACT The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species’ diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

Convergent evolution of gene expression in two high-toothed stickleback populations

“…Following a water rinse, fish were cleared in 0.25% KOH, 50% glycerol for 2–3 weeks. Branchial skeletons were dissected as previously described [ 64 ]. Pharyngeal teeth were quantified with fluorescent illumination using a TX2 filter on a Leica DM2500 microscope.…”

“…Late juvenile stage female sticklebacks (SL ~40mm) were euthanized in 0.04% Tricaine. Dissected [ 64 ] bilateral ventral pharyngeal tooth plates were placed into 500μl TRI reagent, then incubated at room temperature for 5 minutes. Following addition of 100μl of chloroform, a further 10 minute incubation and centrifugation, the aqueous layer was extracted.…”

Convergent evolution of gene expression in two high-toothed stickleback populations

“…Late juvenile stage female sticklebacks (SL ~40mm) were euthanized in 0.04% Tricaine. Dissected [64] bilateral ventral pharyngeal tooth plates were placed into 500µl TRI reagent, then incubated at room temperature for 5 minutes. Following addition of 100µl of chloroform, a further 10 minute incubation and centrifugation, the aqueous layer was extracted.…”

Early development and replacement of the stickleback dentition

“…Fish were rinsed in water and cleared in 50% glycerol, 0.25% KOH. Dissection and mounting was performed as previously described (Miller et al, ; Ellis and Miller, ). Brightfield images were taken on a Leica DM2500 except Figure B which was taken on a Leica M165 stereomicroscope.…”