Center for Quantitative Fisheries Ecology

Protocol

Preparation of Otolith TransverseThin-Sections for Age Estimation of

BluefishPomatomus saltatrix

Copyright c© ∼ Center for Quantitative Fisheries Ecology ∼ 2015

Contents

Equipment and Supplies 1

Introduction 3

Structure of whole sagittal otoliths 3

Preperation for sectioning 4

Sectioning otoliths 6

Mounting otolith sections 8

Storing otolith thin sections 9

Literature Cited 9

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Equipment and Supplies

Item Model/Source Number/UseLeica MZ 95 or Leica MZ12Stereo-microscope with transmittedlight source and polarizing filter

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Buehler R© IsoMetTM low-speed saw Model number11-1280-160 1

Flanges, steel 6.03 cm diameter 2

Spacer, steel 0.5 mm thickness, 6.03cm diameter 1

Norton R© Diamond Grinding Wheel

1A1 3 x 0.006 x 1/2"ME120928,M3D220-N75M99-1/8,UPC/Cup:69014192342

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Allen wrench 1/8 in. 125 gram weights Buehler/1180S33 1-2

Water for IsoMetTMlubricant tray approx. 300ml,unfiltered water

Barnstead 1400 Thermolynefurnace VWR 30605-022 1

Microscope slide [1 inch x 3 inchesx 1.2 mm] VWR 48318-0

1 per otolith plus 4-6extras for "sectioningslide"

Microscope slide storage box VWR 28511-012 1 per 100 otolithsAluminum slide tray VWR 48467 1 per 20 otolithsPorcelain Color Plate VWR 53636-105 1 per 12 otolithsBarnes glass eye dropper anddropper bottle VWR 14216-246 1

Dissecting forceps VWR 82027-398 1 fine point, 1 broad tip

Cordless precision engraver General tools andinstruments 505 1

Metal Spatula General store brand 1Flo-Texx R© liquid cover slip Lerner Laboratories 1 quart

Kimwipes R© Delicate Task Wipersor VWR Light-Duty Tissue Wipers

1 box per work stationfor any spilled orFlo-Texx R©

CQFE bluefish otolith storage box,cardboard

enclosed with labeledcoin envelopes andmicrotubes containingindividual fish’s otoliths

Sharpie R© Ultra Fine PointPermanent Marker 1

Continued on next page

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Table Continued from previous pageItem Model/Source Number/Use

Hard-Part Processing Log

lists selected hard-partsto be processed,categorizedalphabetically byspecies

Silicon baking tray Fresh ware 24-cavity 1West System R© 105-B 3.7 literWest System R© 206-B 814 mlSmall paper Dixie R© cup 3 oz.

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Introduction

The following is a protocol for the prepa-ration of sagittal otolith transverse cross-sections from bluefish (Pomatomus salta-trix ) for age determination. This protocolis to be used after the protocol for collect-ing biometric data and extracting wholesagittal otoliths. We will first provide abrief description of the structure of wholesagittal otoliths from bluefish; describe theprocesses of mounting and sectioning theseotoliths, and mounting and storing thecompleted transverse cross-sections for agedetermination.

Structure of whole sagittalotoliths

Three pairs of otoliths (sagitta, lapil-lus, and asteriscus) are located withinthe vestibluar apparatus of typical Teleostspecies (Figure 1). They all play impor-tant roles in the sensory systems of thesefishes for mechanoreception and mainte-nance of equilibrium in their environment.The sagitta is the largest of the three andused for ageing finfish.

Bluefish have small otoliths relative to theirbody size. Unlike Sciaenids, their saggitaedo not have a unique, tadpole-shaped sul-cus acusticus (sulcus or sulcal groove), but,rather a sulcal groove that extends from theposterior edge to the anterior edge (Figure2). For purpose of this protocol, te sagit-tal otoliths will be referred to simply as"otoliths".

As in all finfishes, bluefish otoliths areformed through the process of biomineral-ization: specifically, the extracellular crys-tallization of calcium carbonate, primarilyaragonite onto an organic matrix composedof a keratin-like protein called "otolin"(Panfili et al. 2002).

Figure 1: Position of otoliths within thevestibular apparatus of typical Teleost species,e.g. bluefish (Secor et al. 1992).

Figure 2: Extracted left and right sagittalotoliths of bluefish labeled to illustrate orien-tation and basic structure.

Otolith formation begins early in the devel-opment of a fish, typically surrounding thehatch-date of the larvae. The initial struc-ture that is mineralizied is called the pri-mordium, or primordia, which fuses to formthe otolith core. This core is the founda-tion on which all new otolith growth occurs.Concentric layers of protein and calciumcarbonate matrix accrete outward from thecore throughout the lifetime of the fish, re-sulting in a structure that is comparable tothat of an onion.

Within the otolith matrix, aragonite is pre-cipitated at varying rates throughout each

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year. Periods of slower growth in the fish,e.g., colder seasons of the year, are charac-terized by densely-packed precipitate. Thecore and opaque layers of the otolith, as vis-ible through transmitted light, respresentsuch growth. Periods of faster growth inthe fish, e.g., warmer seasons of the year,involve less-dense, compacted mineraliza-tion of the precipitate and are indicatedby translucent layers of the matrix whenviewed in transmitted light. The collectionof successive opaque and translucent layerswithin the otolith can be made fully visiblewhen a transverse cross-section (hereafterreferred to as "thin-section") is removedfrom the core region (Figure 3) and viewedthrough a stereomicroscope.

Figure 3: Bluefish otolith, thin-section re-moval, and visible annuli within the section un-der transmitted light.

Each of the opaque and translucent lay-ers within the otolith constitutes an an-nulus, which occurs once per year. Forthe purposes of age determination, only theopaque layers encircling the core are calledannuli; they are counted outwards from thecore towards the outer-edge of the otoliththin-section.

Preparation for sectioning

Begin by turning on the Barnstead/Ther-molyne 1400 Small Benchtop Muffle Fur-nace (hereafter referred to as "oven"). Thetemperature setting should be set to 400◦C (Figure 4).

Figure 4: Barnstead/Thermolyne 1400 SmallBenchtop Muffle Furnace.

To set the temperature:

1. Depress the black button that reads"Push to Set Temp" above it;

2. Turn the "Tempurature" knob until thedisplay reads 400 ◦C;

3. Release the black button.

While the oven is warming, find the speciesand hard-part that has been selected forprocessing from the Hard-Part ProcessingLog. Search for the coin envelope withthe selected Age and Growth ID num-ber within the bluefish otolith storage box.The AGIDs are found on the lower right-hand corner of each envelope.

Figure 5: Tray map.

Then, select the tray map (Figure 5) thatcorresponds with the silicone tray that willbe used to set the otoliths. Record the tray

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Figure 6: Silicon tray and ceramic plate.

number as wells as the AGID of the otolithstarting with the top left cavity going downeach column and ending with the bottomright cavity.

Once the map is appropriately labeled, re-move one otolith from the coin envelopeand place in the cavity that correspondsto the AGID on the map. Do this until thesilicone tray is full.

Transfer the unbaked otoliths one at a timeto the CoorsTek R© Spot Plate (hereafter"ceramic plate"). Be sure to move theotolith in cavity one to well one in the ce-ramic plate, continue with cavity two towell two, etc. (Figure 6). Once all ofthe wells in the ceramic plate have beenfilled, use the metal spatula to pick up thefilled ceramic plate and place it inside theoven. Close the oven door and set thetimer for two minutes. When the timergoes off, remove the ceramic plate withthe metal spatula. Examine the otoliths’color. A light caramel color is ideal for ag-ing. If the color is not caramel enough,put the ceramic plate back into the ovenfor 30 seconds. Sometimes it only takes

one additional 30-second bake; sometimesit takes three to four additional bakes.There should not be any charring or largeblack spots on the otoliths, indicating theotoliths have been over-baked. In somesituations the charring can be removedby scraping it with tweezers. When thelight caramel color is achieved, transfer thebaked otoliths from the ceramic plate to thesilicone tray in the same order the otolithswere originally moved so that the AGIDof the otolith matches the AGID on themap.

The otoliths are now ready to be embeddedin West System R© 2-part epoxy resin (here-after "resin"), part 1 is the 105-B epoxyresin (hereafter "epoxy") and part 2 isthe 206-B slow hardener (hereafter "hard-ener"). The resin is a 5:1 epoxy to hardenerratio. 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 ina 27.5 fl. oz. (814 ml) container (Figure7). To mix the correct ratio you will needone pump of the epoxy and one pump ofthe hardener into a small paper cup (e.g.

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Dixie R© or similar). Be sure to mix theepoxy and hardener well but stir slowly tominimize air bubbles in the mixture. Fillthe silicone cavities until the resin is justabove the top of the otolith, be sure to popor move any air bubbles from around andunderneath the otolith. Leave the resin tocure for 24 hours

Figure 7: Epoxy resin and hardener.

Once the resin is cured, label each blockwith the AGID in permanent marker andremove from the cavities. After removingfrom the cavities, use a dissecting micro-scope to mark the core of each otolith usinga fine-tipped Sharpie R© (Figure 8).

Figure 8: Otolith embedded in resin block withits core marked by Sharpie.

Sectioning otoliths

Before cutting the otolith, make sure thatthe Buehler R© IsoMetTM low speed saw

(hereafter IsoMetTM saw) is set-up cor-rectly. From left to right on the drive-shaftthere should be a shaft spacer and slingerfollowed by an inner flange, a Norton R© di-amond blade (Figure 9), a 0.5 mm spacer,an additional Norton R© diamond blade, anouter flange, and an end cap bushing. Theprevious items are fixed to the drive shaftby a hand-tightened thumb screw (Figure10). The saw’s lubricant pan should befilled with unfiltered water and the speci-men basket should be in place (Figure 11).

Figure 9: Norton R© Diamond Grinding Wheel.

Figure 10: IsoMetTM low speed saw blade in-stallation diagram, showing the order flangeand Norton R© Diamond Grinding Wheel place-ment on the drive shaft. Note, that in our pro-cedure we use a 0.5 mm spacer between twoNorton R© Diamond Grinding Wheels (modi-fied from Buehler R© IsoMetTM Low Speed SawManual).

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Figure 11: IsoMetTM saw lubrication pan di-agram (Buehler R© IsoMetTM Low Speed SawManual).

Place the marked resin block into the chuckof the saw’s support arm (Figure 12), andsecure it using the allen wrench. Once theresin block is secured within the chuck, usethe micrometer (Figure 13) to align thesupport arm and bring the cut line markedon the otolith into position between thetwo Norton R© Diamond Grinding Wheels(hereafter "blades"). The line should runcompletely parallel to both blades, and falldirectly within the 0.5 mm space betweenthem (Figure 14).

Figure 12: Marked resin with otolith embeddedis mounted in the chuck of the saw arm.

When all of the IsoMetTM saw specifica-tions have been met, it is safe to start sec-

Figure 13: IsoMetTM saw weight-balanced di-agram (Buehler R© IsoMetTM Low Speed SawManual).

Figure 14: The black Sharpie R© mark is linedup right between 2 blades.

tioning the otolith. Before moving the sup-port arm and placing the resin block on theblades, start the saw at a speed of 3 or 4.Once the blades have begun spinning, gen-tly move the support arm downward ontothe blades, bring the resin block into con-tact with the blades and allow them to cutat this speed for several seconds. Once theblades have established a groove, bring thesaw speed up to 7 or 8. The blades shouldnow be close to cutting through the otolith,separating the thin-section from the ma-trix. It should take 3 to 5 minutes to com-plete the cut. In some cases the cut willtake longer, maybe over 10 minutes.

The cutting time can be decreased byadding up to 50 grams of weight to theweight shaft at any point during the sec-tioning process. Increasing the saw speed

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will also decrease the amount of time perotolith. Resin is dense and can withstandhigher speeds than the 7 or 8 setting.

Note that additional weight andspeed will increase the risk of damag-ing the saw blades and/or the otolithsections. Technicians must use theirdiscretion, based on personal experi-ence, in sectioning otoliths to main-tain quality and safety.

While the blades are still cutting the resinblock, using a cordless precision engraver,engrave a clean microscope slide (hereafterreferred to as "slide")on the left end withabbreviation name of bluefish (BF), AGID(e.g. 396), VMRC, and the year abbrevia-tion (e.g. 09 for 2009). This slide will beused for mounting the thin-section beingcut.

Monitor the cutting progress and the ad-vancement of the blades through the resinand otolith. From the back of the resinblock, you can view the otolith matrix be-coming thinner as the blades near the backof the resin block. Once the blades cutthrough the entire resin block, stop thesaw. Collect a large piece of resin remain-ing in the chuck and a small piece of resinthat has fallen in the lubricant water, andput them back in the coin envelope fromwhich they came.

Very often, the thin section will stay be-tween the blades. Remove the thumbscrew and the outer flange from the driveshaft. Pull the two blades and spacer off ofthe drive shaft and lay them flat on yourpalm or on the work table. Separate thetop blade from the spacer and the innerblade and search for the thin-section on thespacer side of the blades. If the thin-sectionis not between the blades, then it has falleninto the lubricant water. Pull out the lubri-cant pan, lift the specimen basket out of thewater, search for the thin-section, and col-lect it using tweezers. Using a Kimwipe R©

gently dry the thin section off and place on

the labeled slide.

Mounting otolith thin-section

Before permanently mounting the otoliththin-sections, look at them under the Le-ica stereomicroscope to make sure that thesection includes the core. The sulcal grooveshould meet the core at a precise angle suchthat all annuli can be seen from the originto the edge of the otolith, and sectionedsulcal groove has a "V" shape (Figure 15).

Figure 15: Completed transverse thin-sectionwith a "V" shape of sectioned sulcal groove(positioned as the opening of the "V" down-ward on the slide), indicating that the cut wentthrough the core.

If the sulcal groove and the core does notcome together to form a point, and the sec-tioned sulcal groove looks like a "tornado"shape. Then, the cut was placed too farfrom the ostium (Figure 16).

If the sulcal groove does not come togetherto form a point, and the sectioned sulcalgroove looks like a "dorm" shape. Then,the cut was placed too close to the ostium(Figure 17).

A section with a correctly executed cutshould have no chips or other imperfectionsthat eliminate or obscure views of the core,

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Figure 16: A transverse thin-section with a"tornado" shape of sectioned sulcal groove(showing the opening down here), indicatingthat the cut misses the core and is far awayfrom the ostium.

Figure 17: A transverse thin-section with a"dome" shape of sectioned sulcal groove, in-dicating that the cut misses the core and is tooclose to the ostium.

sulcal groove, or annuli. Sections not meet-ing these specifications must be re-cut. Thesecond section may be obtained from eitherof the two halves of the first otolith (de-pending on where the first section missesthe core), but the second otolith may alsobe used if necessary. When you have ver-ified the section’s quality, place the bestsurface of the thin-section facing upwardsand ensure the section is siting flat on thepreviously engraved slide. Make sure thatthe opening of the "V" shape is positionedfacing the long edge of the slide while theengraved end of the slide is facing to theleft of reader (Figure 18).

When the section is clean and dry, use aneyedropper to put a small amount of Flo-

Texx R© over the section and spread it in acircular motion. Eliminate bubbles withinthe Flo-Texx R© by popping them or mov-ing them away from the thin-section us-ing tweezers. The Flo-Texx R© is used asthe mounting medium to both protect thethin-section and increase its clarity. Placethe completed slides on an aluminum slidetray and allow the Flo-Texx R© to air-dry onthe sections for several hours (until solidi-fied).

Storing otolith thin-sections

Using a fine point black Sharpie R©, writethe abbreviation name of bluefish (BF)and fish ID (396) in the upper right-handcorner on the long side of the slide edge(This step may be done while engravingthe slide). Figure 18 shows the final otoliththin-section slide correctly labeled with ab-breviation of species name, fish ID, VMRC,and collection year. Store the slides ina labeled slide box (Figure 19) with theblack Sharpie R© AGID facing upwards foreasy identification during age determina-tion.

Figure 18: Completed bluefish otolith thin-section, labeled appropriately with speciescode and AGID.

Literature Cited

Buehler R© IsoMetTM Low Speed Saw Man-ual. Model MA111280_16.1 as of6/8/2009.

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Figure 19: Otolith slide storage box, labeledfor bluefish.

Panfili, J., Rontual, H. Troadac, H.,Wright, P. 2002. Manual of Fish Schle-rochronology, pp35.

Secor, D.H., Dean, J.M., and Laban, E.H.1992. Otolith removal and prepara-tion for microstructural examination.In Otolith microstructure examinationand analysis (Stevenson, D.K. and Cam-pana, S.E., ed.).

Photographs

James Davies, Karen Underkof-fler, Christina Morgan, and JessicaGilmore.

Prepared by

James Davies, William Persons, ChristinaMorgan, Hongsheng Liao, CynthiaJones,Steve Bobko, Eric Robillard, KarenUnderkoffler, and Jessica Gilmore.

Production date

September 30, 2015

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