Published: May 7th, 2016
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.
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.
An incredible amount of diversity exists in the head skeleton among vertebrates, especially among fishes. In many cases this diversity facilitates different feeding strategies1-4, and can involve major changes to both external and internal craniofacial patterning. The branchial skeleton is located internally in the throat of a fish and surrounds most of the buccal cavity. The branchial skeleton is comprised of 5 serially homologous segments, the anterior four of which support the gills. Together these five segments function as an interface between fish and their food5. Variation in a multitude of traits including gill rakers....
All fish work was approved by the Institutional Animal Care and Use Committee of the University of California-Berkeley (protocol number R330). Euthanasia was performed using immersion in 0.025% Tricaine-S buffered with 0.1% sodium bicarbonate39. All steps are performed at room temperature.
Note: Perform steps 1.1-1.5 in conical tubes or scintillation vials that can seal tightly and be laid horizontally. Fish do not need to be constantly shaken, but try to mix the solution as of.......
This protocol results in a dissected and flat mounted branchial skeleton (Figure 4) where a variety of important trophic traits can be quantified. From a dorsal view, all rows of gill rakers, all pharyngeal tooth plates, and nearly all branchial bones can be easily visualized and quantified22-24,35,36,38,42. Alizarin Red S also fluoresces on a rhodamine or similar red filter allowing double labeling with other markers (e.g., trans.......
The branchial skeleton is a complex set of bones in the throat of a fish that manipulates, filters, and masticates food items on their way to the esophagus. Many interesting trophic traits including the patterning of gill rakers, pharyngeal teeth, and branchial bones vary across and within species. The majority of these traits are difficult to near impossible to accurately measure with the branchial skeleton in situ (e.g., gill raker length, branchial bone length). This flat-mounting protocol places all.......
This work was funded in part by NIH R01 #DE021475 to CTM and an NSF Graduate Research Fellowship to NAE. Thanks to Miles Johnson for assistance with imaging and Priscilla Erickson for critical reading of the manuscript.....
|Sodium Hydroxide (KOH)
|10% Neutral Buffered Formalin (NBF)
|Alizarin Red S
|Microscope Cover Glasses 22x60mm
|100x10mm Glass Petri Dish
|To dissect samples on
|Sylgard 184 Silicone Elastomer Kit
|184 SIL ELAST KIT 0.5KG
|Can be poured into glass or plastic petri dishes to make dissecting plates
|Scintillation Vials (case of 500)
|Borosilicate Glass with Screw Cap
|Forceps-Dumont #5 Inox (Biologie tip)
|Dumostars are an alternative
|Alternate sizes are available depending on size of sample
|S6E with KL300 LED
|Many other models work nicely, having a flat base helps
|Microcentrifuge Tubes 1.7mL
|Cardboard slide tray
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