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Protocol Preparation of Otolith Transverse Thin-Sections for Age

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Protocol Preparation of Otolith Transverse Thin-Sections for Age Center for Quantitative Fisheries Ecology Protocol Preparation of Otolith Transverse Thin-Sections for Age Estimation of Golden Tilefish Lopholatilus chamaeleonticeps Copyright c ∼ Center for Quantitative Fisheries Ecology ∼ 2016 Contents Equipment and Supplies1 Introduction3 Structure of whole sagittal otoliths3 Preparation for sectioning4 Sectioning otoliths5 Mounting otolith sections7 Storing otolith thin sections8 Literature Cited9 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 Microscope slide [1 inch x 3 inches VWR 48318-0 1 per otolith x 1.2 mm] Microscope slide storage box VWR 28511-012 1 per 100 otoliths Aluminum slide tray VWR 48467 1 per 20 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 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 golden tilefish otolith coin envelopes and storage box, cardboard microtubes containing individual fish’s otoliths Sharpie R Ultra Fine Point 1 Permanent Marker 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 Continued on next page 1 Table Continued from previous page Item Model/Source Number/Use 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 golden tilefish (Lopholatilus chamaeleonticeps) for age determination. This protocol is to be used after the pro- tocol for collecting biometric data and ex- tracting whole sagittal otoliths. We will briefly introduce the structure of whole sagittal otoliths from golden tilefish, and then describe the detailed processes of mounting and sectioning these 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. golden tilefish (Secor et al. 1992). Structure of whole sagittal Otolith formation begins early in the devel- otoliths opment of a fish, typically surrounding the hatch-date of the larvae. The initial struc- ture that is mineralizied is called the pri- Three pairs of otoliths (sagitta, lapil- mordium, or primordia, which fuses to form lus, and asteriscus) are located within the otolith core. This core is the founda- the vestibluar apparatus of typical Teleost tion on which all new otolith growth occurs. species (Figure1). They all play impor- Concentric layers of protein and calcium tant roles in the sensory systems of these carbonate matrix accrete outward from the fishes for mechanoreception and mainte- core throughout the lifetime of the fish, re- nance of equilibrium in their environment. sulting in a structure that is comparable to The sagitta is the largest of the three and that of an onion. used for ageing finfish. Unlike Sciaenids, golden tilefish saggitae Within the otolith matrix, aragonite is pre- do not have a unique, tadpole-shaped sul- cipitated at varying rates throughout each cus acusticus (sulcus or sulcal groove), but, year. Periods of slower growth in the fish, rather a sulcal groove that extends from i.e., colder seasons of the year, are charac- the posterior edge to the anterior edge (Fig- terized by densely-packed precipitate. The ure2). For purposes of this protocol, the core and opaque layers of the otolith, as vis- sagittal otoliths will be referred to simply ible through transmitted light, respresent as "otoliths". such growth. Periods of faster growth in the fish, i.e., warmer seasons of the year, As in all finfishes, golden tilefish otoliths involve less-dense, compacted mineraliza- are formed through the process of biomin- tion of the precipitate and are indicated eralization: specifically, the extracellular by translucent layers of the matrix when crystallization of calcium carbonate (pri- viewed in transmitted light. The collection marily aragonite) onto an organic matrix of successive opaque and translucent layers composed of a keratin-like protein called within the otolith can be made fully visible "otolin" (Panfili et al. 2002). when a transverse cross-section (hereafter 3 envelope with the selected Age and Growth ID number within golden tilefish otolith storage box. The AGIDs are found on the lower right-hand corner of each enve- lope. Then, select the tray map (Figure4) that corresponds with the silicone tray that will be used to set the otoliths. Record the tray number as wells as the AGID of the otolith starting with the top left cavity going down each column and ending with the bottom right cavity. Figure 2: Extracted left and right sagittal otoliths of golden tilefish labeled to illustrate orientation and basic structure. referred to as "thin-section") is removed from the core region (Figure3) and viewed through a stereomicroscope. Figure 4: Tray map. Figure 3: Golden tilefish otolith, thin-section Once the map is appropriately labeled, re- removal, and visible annuli within the section move one otolith from the coin envelope under transmitted light. and place in the cavity that corresponds to the AGID on the map. Do this until the Each of the opaque and translucent lay- silicone tray is full. ers within the otolith constitutes an an- The otoliths are now ready to be embedded nulus, which occurs once per year. For in West System R 2-part epoxy resin (here- the purposes of age determination, only the after "resin"), part 1 is the 105-B epoxy opaque layers encircling the core are called resin (hereafter "epoxy") and part 2 is annuli; they are counted outwards from the the 206-B slow hardener (hereafter "hard- core towards the outer-edge of the otolith ener"). The resin is a 5:1 epoxy to hardener thin-section. ratio. More specifically, to achieve the re- quired ratio, the epoxy is in a 126.6 fl. oz. (3.7 liter) container and the hardener is in Preparation for sectioning a 27.5 fl. oz. (814 ml) container (Figure 5). To mix the correct ratio you will need Find the species and hard-part that has one pump of the epoxy and one pump of been selected for processing from the Hard- the hardener into a small paper cup (e.g. Part Processing Log. Search for the coin Dixie R or similar). Be sure to mix the 4 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 (Figure7), a 0.5 mm spacer, underneath the otolith. Leave the resin to an additional Norton R diamond blade, an cure for 24 hours outer flange, and an end cap bushing. The previous items are fixed to the drive shaft by a hand-tightened thumb screw (Figure 8). The saw’s lubricant pan should be filled with unfiltered water and the spec- imen basket should be in place (Figure9). Figure 5: Epoxy resin and hardener. Once the resin is cured, label each block with the AGID in permanent marker and remove from the cavities. After removing from the cavities, use a dissecting micro- Figure 7: Norton R Diamond Grinding Wheel. scope mark the core of each otolith using a fine-tipped Sharpie R (Figure6). Figure 6: Otolith embedded in resin block with its core marked by Sharpie. Figure 8: IsoMetTM low speed saw blade in- stallation diagram, showing the order flange and Norton R Diamond Grinding Wheel place- Sectioning otoliths ment on the drive shaft. Note, that in our pro- cedure we use a 0.5 mm spacer between two Norton R Diamond Grinding Wheels (modi- Before cutting the otolith, make sure that fied from Buehler R IsoMetTM Low Speed Saw the Buehler R IsoMetTM low speed saw Manual). 5 TM Figure 9: IsoMetTM saw lubrication pan di- Figure 11: IsoMet saw weight-balanced di- R TM agram (Buehler R IsoMetTM Low Speed Saw agram (Buehler IsoMet Low Speed Saw Manual). Manual). Place the marked resin block into the chuck of the saw’s support arm (Figure 10),and secure it using the allen wrench. Once the resin block is secured within the chuck, use the micrometer (Figure 11) to align the support arm and bring the cut line marked on the otolith into position between the two Norton R Diamond Grinding Wheels (hereafter "blades"). The line should run completely parallel to both blades, and fall directly within the 0.5 mm space between them (Figure 12). Figure 12: The black Sharpie R mark is lined up right between 2 blades. port arm down and placing the resin block on to the blades, start the saw at a speed of 3 or 4.
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