Protocol Preparation of Otolith Transverse Thin-Sections ......UPC/Cup: 69014192342 2 Allenwrench...

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Center for Quantitative Fisheries Ecology Protocol Preparation of Otolith Transverse Thin-Sections for Age Estimation of Weakfish Cynoscion regalis Copyright c Center for Quantitative Fisheries Ecology 2015

Transcript of Protocol Preparation of Otolith Transverse Thin-Sections ......UPC/Cup: 69014192342 2 Allenwrench...

Page 1: Protocol Preparation of Otolith Transverse Thin-Sections ......UPC/Cup: 69014192342 2 Allenwrench 1/8in. 1 25gramweights Buehler/1180S33 1-2 WaterforIsoMetTMlubricanttray approx. 300ml,

Center for Quantitative Fisheries Ecology

Protocol

Preparation of Otolith TransverseThin-Sections for Age Estimation of

WeakfishCynoscion regalis

Copyright c© ∼ Center for Quantitative Fisheries Ecology ∼ 2015

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Contents

Equipment and Supplies 1

Introduction 3

Mounting Otoliths for Sectioning 4

Sectioning otoliths 5

Mounting otolith thin-sections 7

Storing otolith thin-sections 8

Literature Cited 8

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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

Ceramic top hot plateModel number VWR82026-752 115V,1000W, 7x7"

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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 otolithsBarnes glass eye dropper anddropper bottle VWR 14216-246 1

Stender dish with lid VWR WLS 26155-C 1Dissecting forceps VWR 82027-398 1 fine point, 1 broad tip

Cordless precision engraver General tools andinstruments 505 1

Crystalbond 509-S AREMCO ProductsInc. 1/4 stick

Applicator stick 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©

Razor blade

1 for removingremnants of CrystalBond from microscopeslides post-sectioning

Continued on next page

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Table Continued from previous pageItem Model/Source Number/Use

CQFE weakfish otolith storage box,cardboard

enclosed with labeledcoin envelopes andmicrotubes containingindividual fish’s otoliths

Sharpie R© Ultra Fine PointPermanent Marker 1

Hard-Part Processing Log

lists selected hard-partsto be processed,categorizedalphabetically byspecies

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Introduction

The following is a protocol for the prepa-ration of sagittal otolith transverse cross-sections from weakfish (Cynoscion regalis)for age determination. This protocol isto be used after the protocol for collect-ing biometric data and extracting wholesagittal otoliths. The protocol will firstbriefly introduce the structure of wholesagittal otoliths from weakfish, and thendescribe the detailed processes of mountingand sectioning the otoliths, and mountingand storing the completed transverse cross-sections for age determination.

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.

Like all sciaenids, weakfish have extremelylarge sagittal otoliths relative to their bodysize. Their saggitae have a unique, tadpole-shaped sulcus acusticus (sulcus or sulcalgroove) with an enlarged ostium anteriorand tail-like cauda posterior (Figure 2).For purposes of this protocol, the sagit-tal otoliths will be referred to simply as"otoliths".

As in all finfishes, weakfish otoliths areformed through biomineralization: specif-ically, the extracellular crystallization ofcalcium carbonate (primarily aragonite)onto an organic matrix which is 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. weakfish (Secor et al. 1992).

Otolith formation begins early in the devel-opment of a fish, typically at the hatch-dateof the larvae. The initial structure that ismineralizied is called the primordium, orprimordia, which fuses to form the otolithcore. This core is the foundation on whichall new otolith growth occurs. Concentriclayers of the protein and calcium carbon-ate matrix accrete, or grow, outward fromthe core throughout the lifetime of the fish.This results in a structure that is compa-rable to that of an onion.

Within the otolith matrix, aragonite is pre-cipitated at varying rates throughout eachyear. Periods of slower growth in the fish,i.e., colder seasons of the year, are charac-terized by densely-packed precipitate. Thecore and opaque layers of the otolith, visi-ble under transmitted light, represent suchgrowth. Periods of faster growth in the fish,i.e., warmer seasons of the year, involveless-densely compacted mineralization ofthe precipitate and are seen as translucentlayers of the matrix when viewed in trans-mitted light.

The collection of successive opaque andtranslucent layers within the otolith canbe made fully visible when a transverse

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Figure 2: Extracted left and right sagittalotoliths of weakfish labeled to illustrate orien-tation and basic structure (The otoliths are po-sitioned at the proximal facing front).

cross-section (hereafter referred to as "thin-section") is removed from the core region(Figure 3) and viewed through a stereomi-croscope. Each of the opaque and translu-cent layers within the otolith constitutes anannulus, which occurs once per year. For

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

the purposes of age determination, only theopaque layers encircling the core are calledannuli. They are counted from the coretowards the outer-edge of the otolith thin-section.

Mounting Otoliths for Sec-tioning

First turn on the hot plate. Each of thehot plates have a recommended tempera-ture setting marked previously with blackSharpie R©. For example, the VWR brandhot plate setting is marked at ∼1.5 and theCorning at ∼3.5.

Place 14 stick of Crystalbond R© 509 (here-

after referred to as "Crystalbond") into aStender dish. Place the dish on the hotplate surface. While the hot plate is warm-ing to its recommended temperature, re-fer to the Hard-Part Processing Log andidentify the hard-part that has been se-lected for processing. Remove the coin en-velope with the appropriate weakfish Ageand Growth ID number (AGID) from theCQFE weakfish otolith storage box. TheAGID is located on the lower right-handcorner of each envelope.

When the hot plate has reached its propertemperature and the Crystalbond has be-come easy to manipulate, place a micro-scope slide (hereafter referred to as "slide")on the hot plate surface. Using an ap-plicator stick, evenly spread the semi-liquid Crystalbond on the slide. Place an-other slide on top of the Crystalbond cov-ered slide, makeing a slide-Crystalbond-slide sandwich. Press the slides togetherwith the applicator stick to evenly dis-tribute the semi-liquid Crystalbond. Here-after, this will be referred to as a "section-ing slide".

Using tweezers, set the sectioning slideaside on the table to cool to room temper-ature. Remove an otolith from the coin en-velope. When the sectioning slide is cool tothe touch, use the applicator stick to placethe semi-liquid Crystalbond in the center ofthe third quarter of the sectioning slide tomake a base of Crystalbond. The amountof the Crystalbond base needed is roughly

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equal to the volume of the otolith that willbe sectioned.

Mount the otolith, with the proximal fac-ing up, half way into the Crystalbond base.Make sure that the dorsal and ventral sidesof the otolith lie parallel to the long edgeof the slide and perpendicular to the shortedges. Apply a layer of the semi-liquidCrystalbond on top of the tips of the ante-rior and posterior of the otolith to securethem in place. Set the sectioning slide,with the mounted otolith, on the table un-til it is cool and the otolith is firmly lockedin the Crystalbond.

With a pencil, draw a straight line on theotolith to indicate the path the saw bladeswill follow (This step may be done beforemounting the otolith on the slide). Thelines should set-up the cut so that the po-sition of the core is found within the thin-section to be removed from the otolith.The core is located at the posterior por-tions of the sulcal groove and close to theostium. Two black dash lines are markedin Figure 3 and a pencil mark is placed onotolith in Figure 4 to indicate this position.The line should be perpendicular to the sul-cal groove and just next to, but not over-lapping, the tadpole head-like lobes of theostium. When sectioning, the saw bladesshould fall on either side of the pencil line.

Figure 4: Weakfish otolith marked with cut linemounted on a sectioning slide.

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 5), 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 (Figure6). The saw’s lubricant pan should befilled with unfiltered water and the spec-imen basket should be in place (Figure 7).

Figure 5: Norton R© Diamond Grinding Wheel.

Place the sectioning slide with the wholeotolith in crystalbond and marked withpencil into the chuck of the saw’s supportarm (Figure 8), gently secure it using theallen wrench. Once the slide is securedwithin the chuck, use the micrometer (Fig-ure 9) to align the support arm and bringthe cut line marked on the otolith into po-sition between the two Norton R© DiamondGrinding Wheels (hereafter referred to as"blades"). The line should run completelyparallel to both blades, and fall directlywithin the 0.5 mm space between them(Figure 10).

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Figure 6: 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).

Figure 7: IsoMetTM saw lubrication pan di-agram (Buehler R© IsoMetTM Low Speed SawManual).

When all of the IsoMetTM saw specifica-tions have been met, it is safe to start sec-tioning the otolith. Before moving the sup-port arm and marked otolith down on tothe blades, start the saw at a speed of 3or 4. Once the blades have begun spin-ning, gently move the support arm down-ward onto the blades, bringing the securedotolith into contact with the blades. Allowthe blades to cut at this speed for severalseconds. Once the blades have establisheda groove, bring the saw speed up to 7 or8.

Figure 8: The sectioning slide with otolith se-cured in crystalbond is mounted in the chuckof the saw arm.

Figure 9: IsoMetTM saw weight-balanced di-agram (Buehler R© IsoMetTM Low Speed SawManual).

The cutting time varies with otlith size, sawspeed, and weight. The cutting time canbe decreased by adding up to 50 grams ofweight to the weight shaft at any point dur-ing the sectioning process. Increasing thesaw speed will also decrease the amountof time per otolith. weakfish otoliths arefairly robust and can withstand higherspeeds than the 7 or 8 setting.

Note that additional weight andspeed could increase the risk ofdamaging the saw blades and/orthe otolith sections. Techniciansmust use their discretion, basedon personal experience, in section-ing otoliths to maintain quality and

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Figure 10: The pencil mark is lined up rightbetween 2 blades.

safety.

While the blades are still cutting theotolith, using a cordless precision engraver,engrave a clean slide on the left end withthe abbreviation name of weakfish (CRK),AGID (e.g. 002), VMRC, and the year ab-breviation (e.g. 14 for 2014). This slidewill be used for mounting the thin-sectionbeing cut.

Monitor the cutting progress of the bladesthrough the otolith. From the back of theslide, you can view the otolith matrix be-coming thinner as the blades near the Crys-talbond base and the underlying glass ofthe sectioning slide. When you see that theblades have gone completely through thematrix and are visible on either side of thethin-section being removed, carefully raisethe support arm from the saw blades andturn off the IsoMetTM saw.

The just cut thin-section should remainsuspended in the Crystalbond between theseparated halves of the otolith. Use tweez-ers to carefully remove the thin-sectionfrom between the two halves. If the thin-section is not located between two halves ofthe otlith, the thin-section either is stuckbetween the blades or has fallen into thelubricant water. In the first situation, re-move the thumb screw and the outer flangefrom the drive shaft. Pull the two bladesand spacer off of the drive shaft and lay

them flat on your palm or on the work ta-ble. Separate the top blade from the spacerand the inner blade and search for the thin-section on the spacer side of the blades. Inthe second situation, pull out the lubricantpan, lift the specimen basket out off water,search for the thin-section, and collect itusing tweezers.

Mounting otolith thin-section

Before permanently mounting the otoliththin-sections, look at them under the Le-ica stereomicroscope to make sure that thesection must include the core. The sulcalgroove should meet the core at a precise an-gle such that all annuli can be seen from theorigin to the edge of the otolith, and sec-tioned sulcal groove has a "V" shape (Fig-ure 11).

Figure 11: 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 do notcome together to form a point, and the sec-tioned sulcal groove looks like a "tornado"shape, then, the cut was placed too far fromthe ostium (Figure 12).

If the sulcal groove does not come togetherto form a point, and the sectioned sulcalgroove looks like a "dome" shape, then, thecut was placed too close to the ostium (Fig-ure 13).

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Figure 12: 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 13: 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.

A section with a correctly executed cutshould have no chips or other imperfectionsthat eliminate or obscure views of the core,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 veri-fied the section’s quality, place the best sur-face of the thin-section facing upwards andensure the section is siting flat on the slideengraved previously. Make sure that theopening of the "V" shape is positioned fac-ing the long edge of the slide while the en-graved end faces to the left of reader.

When the section is clean and dry, protectit with Flo-Texx R©, a liquid cover slip. Usean eyedropper to put a small amount ofFlo-Texx R© over the section and spread it

in a circular motion. Eliminate bubbleswithin the Flo-Texx R© by popping them ormoving them away from the thin-sectionusing tweezers. The Flo-Texx R© is used asthe mounting medium to both protect thethin-section and increase its clarity. Placethe completed slide on an aluminum slidetray and allow the Flo-Texx R© to air-dry onthe thin-section for several hours (until so-lidified).

Now that the quality of the section hasbeen verified and the slide has been placedaside to dry, use a razor blade to removethe two halves of the otolith with the sur-rendering crystalbond off from the section-ing slide. Clean the crystalbond from thetwo halves of the otolith and the sectioningslide, then, collect and put it back into theStender dish for re-usage. Place the twohalves of the otolith back into the coin en-velope from which they came, and re-usethe sectioning slide for next otolith.

Storing otolith thin-sections

Using a fine point black Sharpie R©, writethe abbreviation name of weakfish (CRK)and fish ID (002) in the upper right-handcorner (opposite to the engraved end) onthe long side of the slide edge (This stepmay be done while engraving the slide).Figure 14 shows the final otolith thin-section slide correctly labeled with abbre-viation of species name, fish ID, VMRC,and collection year. Store the slides ina labeled slide box (Figure 15) with theblack Sharpie R© AGID facing upwards foreasy identification during age determina-tion.

Literature Cited

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

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Figure 14: Completed weakfish otolith thin-section, labeled appropriately with speciescode and AGID.

Figure 15: Otolith slide storage box, labeledfor weakfish.

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.).

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

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 28, 2015

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