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Striped Bass Center for Quantitative Fisheries Ecology Protocol Preparation of Otolith Transverse Thin-Sections for Age Estimation of Striped bass Morone saxatilis Copyright c ∼ Center for Quantitative Fisheries Ecology ∼ 2015 Contents Equipment and Supplies1 Introduction3 Structure of whole sagittal otoliths3 Preperation for sectioning4 Sectioning otoliths6 Mounting otolith sections8 Storing otolith thin sections9 Literature Cited 10 i Equipment and Supplies Item Model/Source Number/Use Leica MZ 95 or Leica MZ12 Stereo-microscope with transmitted 1 light source and polarizing filter Model number Buehler R IsoMetTM low-speed saw 1 11-1280-160 Flanges, steel 6.03 cm diameter 2 0.5 mm thickness, 6.03 Spacer, steel 1 cm diameter 1A1 3 x 0.006 x 1/2" ME120928, Norton R Diamond Grinding Wheel M3D220-N75M99-1/8, 2 UPC/Cup: 69014192342 Allen wrench 1/8 in. 1 25 gram weights Buehler/1180S33 1-2 approx. 300ml, Water for IsoMetTMlubricant tray unfiltered water Barnstead 1400 Thermolyne VWR 30605-022 1 furnace 1 per otolith plus 4-6 Microscope slide [1 inch x 3 inches VWR 48318-0 extras for "sectioning x 1.2 mm] slide" Microscope slide storage box VWR 28511-012 1 per 100 otoliths Aluminum slide tray VWR 48467 1 per 20 otoliths Porcelain Color Plate VWR 53636-105 1 per 12 otoliths Barnes glass eye dropper and VWR 14216-246 1 dropper bottle Dissecting forceps VWR 82027-398 1 fine point, 1 broad tip General tools and Cordless precision engraver 1 instruments 505 Metal Spatula General store brand 1 Flo-Texx R liquid cover slip Lerner Laboratories 1 quart 1 box per work station Kimwipes R Delicate Task Wipers for any spilled or or VWR Light-Duty Tissue Wipers Flo-Texx R enclosed with labeled CQFE striped bass otolith storage coin envelopes and box, cardboard microtubes containing individual fish’s otoliths Sharpie R Ultra Fine Point 1 Permanent Marker Continued on next page 1 Table Continued from previous page Item Model/Source Number/Use lists selected hard-parts to be processed, Hard-Part Processing Log categorized alphabetically by species Silicon baking tray Fresh ware 24-cavity 1 West System R 105-B 3.7 liter West System R 206-B 814 ml Small paper Dixie R cup 3 oz. 2 Introduction The following is a protocol for the prepa- ration of sagittal otolith transverse cross- sections from striped bass (Morone sax- atilis) for age determination. This pro- tocol is to be used after the protocol for collecting biometric data and extract- ing whole sagittal otoliths. The protocol will first briefly introduce the structure of whole sagittal otoliths from striped bass, and then describe the detailed processes of mounting and sectioning the otoliths, and mounting and storing the completed transverse cross-sections for age determina- Figure 1: Position of otoliths within the tion. vestibular apparatus of typical Teleost species, e.g., striped bass (Secor et al. 1992). Structure of whole sagittal otoliths Three pairs of otoliths (sagitta, lapil- lus, and asteriscus) are located within the vestibluar apparatus of typical Teleost species (Figure1). They all play impor- tant roles in the sensory systems of these fishes for mechanoreception and mainte- nance of equilibrium in their environment. The sagitta is the largest of the three and used for ageing finfish. Figure 2: Extracted left and right sagittal otoliths of striped bass labeled to illustrate ori- Striped bass have small otoliths relative to entation and basic structure. their body size. Unlike Sciaenids, their sag- gitae do not have a unique, tadpole-shaped sulcus acusticus (sulcus or sulcal groove), Otolith formation begins early in the devel- but, rather a sulcal groove that extends opment of a fish, typically surrounding the from the posterior edge to the anterior edge hatch-date of the larvae. The initial struc- (Figure2). For purposes of this protocol, ture that is mineralizied is called the pri- the sagittal otoliths will be referred to sim- mordium, or primordia, which fuses to form ply as "otoliths". the otolith core. This core is the founda- tion on which all new otolith growth occurs. As in all finfishes, striped bass otoliths are Concentric layers of protein and calcium formed through biomineralization: specif- carbonate matrix accrete outward from the ically, the extracellular crystallization of core throughout the lifetime of the fish, re- calcium carbonate (primarily aragonite) sulting in a structure that is comparable to onto an organic matrix composed of a that of an onion. keratin-like protein called "otolin" (Panfili et al. 2002). Within the otolith matrix, aragonite is pre- 3 cipitated at varying rates throughout each nace (hereafter referred to as "oven"). The year. Periods of slower growth in the fish, temperature setting should be set to 400 i.e., colder seasons of the year, are charac- ◦C (Figure4). terized by densely-packed precipitate. The core and opaque layers of the otolith, as vis- ible through transmitted light, respresent such growth. Periods of faster growth in the fish, i.e., warmer seasons of the year, involve less-dense, compacted mineraliza- tion of the precipitate and are indicated by translucent layers of the matrix when viewed in transmitted light. The collection of successive opaque and translucent layers within the otolith can be made fully visible when a transverse cross-section (hereafter Figure 4: Barnstead/Thermolyne 1400 Small referred to as "thin-section") is removed Benchtop Muffle Furnace. from the core region (Figure3) and viewed through a stereomicroscope. To set the temperature: 1. Depress the black button that reads "Push to Set Temp" above it; 2. Turn the "Tempurature" knob until the display reads 400 ◦C; 3. Release the black button. While the oven is warming, find the species and hard-part that has been selected for processing from the Hard-Part Processing Log. Search for the coin envelope with the Figure 3: Striped bass otolith, thin-section re- selected Age and Growth ID number within moval, and visible annuli within the section un- der transmitted light. the striped bass otolith storage box. The AGIDs are found on the lower right-hand corner of each envelope. Each of the opaque and translucent lay- ers within the otolith constitutes an an- nulus, which occurs once per year. For the purposes of age determination, only the opaque layers encircling the core are called annuli; they are counted from the core to- wards the outer-edge of the otolith thin- section. Preparation for sectioning Begin by turning on the Barnstead/Ther- molyne 1400 Small Benchtop Muffle Fur- Figure 5: Tray map. 4 Figure 6: Silicon tray and ceramic plate. Then, select the tray map (Figure5) that ing. If the color is not caramel enough, corresponds with the silicone tray that will put the ceramic plate back into the oven be used to set the otoliths. Record the tray for 30 seconds. Sometimes it only takes number as wells as the AGID of the otolith one additional 30-second bake; sometimes starting with the top left cavity going down it takes three to four additional bakes. each column and ending with the bottom There should not be any charring or large right cavity. black spots on the otoliths, indicating the otoliths have been over-baked. In some Once the map is appropriately labeled, re- situations the charring can be removed move one otolith from the microtube in the by scraping it with tweezers. When the coin envelope and place in the cavity that light caramel color is achieved, transfer the corresponds to the AGID on the map. Do baked otoliths from the ceramic plate to the this until the silicone tray is full. silicone tray in the same order the otoliths Transfer the unbaked otoliths one at a time were originally moved so that the AGID to the CoorsTek R Spot Plate (hereafter of the otolith matches the AGID on the "ceramic plate"). Be sure to move the map. otolith in cavity one to well one in the ce- ramic plate, continue with cavity two to The otoliths are now ready to be embedded well two, etc. (Figure6). Once all of in West System R 2-part epoxy resin (here- the wells in the ceramic plate have been after "resin"), part 1 is the 105-B epoxy filled, use the metal spatula to pick up the resin (hereafter "epoxy") and part 2 is filled ceramic plate and place it inside the the 206-B slow hardener (hereafter "hard- oven. Close the oven door and set the ener"). The resin is a 5:1 epoxy to hardener timer for two minutes. When the timer ratio. More specifically, to achieve the re- goes off, remove the ceramic plate with quired ratio, the epoxy is in a 126.6 fl. oz. the metal spatula. Examine the otoliths’ (3.7 liter) container and the hardener is in color. A light caramel color is ideal for ag- a 27.5 fl. oz. (814 ml) container (Figure 5 7). To mix the correct ratio you will need Sectioning otoliths one pump of the epoxy and one pump of the hardener into a small paper cup (e.g. Before cutting the otolith, make sure that R Dixie or similar). Be sure to mix the the Buehler R IsoMetTM low speed saw epoxy and hardener well but stir slowly to (hereafter IsoMetTM saw) is set-up cor- minimize air bubbles in the mixture. Fill rectly. From left to right on the drive-shaft the silicone cavities until the resin is just there should be a shaft spacer and slinger above the top of the otolith, be sure to pop followed by an inner flange, a Norton R di- or move any air bubbles from around and amond blade (Figure9), a 0.5 mm spacer, underneath the otolith.
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