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Otolith Aging and Analysis Otolith Aging and Analysis Presented by Presented by Bill Stewart Bill Stewart Arizona State University Arizona State University Computational Biology Program Computational Biology Program Arizona Game and Fish Department Arizona Game and Fish Department

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Page 1: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Otolith Aging and AnalysisOtolith Aging and Analysis

Presented byPresented by Bill StewartBill Stewart

Arizona State University Arizona State University Computational Biology ProgramComputational Biology Program

Arizona Game and Fish DepartmentArizona Game and Fish Department

Page 2: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Project GoalsProject Goals Create a program that calculates a fish's Create a program that calculates a fish's

age by anaylzing digital images of it's age by anaylzing digital images of it's otoliths.otoliths.

Page 3: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Structures used to age fishStructures used to age fish

ScalesScales

BonesBones

Fin RaysFin Rays

OtolithsOtoliths

Page 4: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

OtolithsOtoliths

Otoliths are small calcified structures used for balance and hearing in fish.

Page 5: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Otolith StructureOtolith Structure

Each fish has three Each fish has three pairspairs

– SagittaeSagittae

– LapilliLapilli

– AsterisciiAsteriscii

Different shapes and Different shapes and sizessizes

Page 6: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Formation of AnnuliFormation of Annuli

Otoliths have continuous growth. So as new material is added to the Otoliths have continuous growth. So as new material is added to the outside surface the older material is preserved providing a record of outside surface the older material is preserved providing a record of the fish's life.the fish's life.

Otoliths form daily rings which during periods of slow growth pile up Otoliths form daily rings which during periods of slow growth pile up and form annular rings.and form annular rings.

Page 7: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

How Biologists Use OtolithsHow Biologists Use Otoliths

Temperature history Temperature history (Patterson et al. 1993)(Patterson et al. 1993)

Anadromy Anadromy (Secor 1992)(Secor 1992)

Migration Pathway Migration Pathway (Thresher et al. 1994)(Thresher et al. 1994)

Stock Identification Stock Identification (Edmonds et al. 1989)(Edmonds et al. 1989)

Used as a natural tag Used as a natural tag (Campana et al. 1995)(Campana et al. 1995)

Age ValidationAge Validation (many publications)(many publications)

Page 8: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Otolith PreparationOtolith Preparation

Step one: Mounting on slideStep one: Mounting on slide

Step two: Grinding until translucentStep two: Grinding until translucent

(Very time consuming process.)

Page 9: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Examples of imagesExamples of images

White Bass Age 2

White Bass Age 4

Largemouth Bass Age 3

White Bass Age 3

Page 10: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

MatlabMatlab

Image enhancementImage enhancement

Methods for counting and measuring distance Methods for counting and measuring distance between annulibetween annuli

BackcalculationsBackcalculations

Three Parts

Page 11: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Image EnhancementImage Enhancement

Rgb2gray intensity imageRgb2gray intensity image

Page 12: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Image EnhancementImage Enhancement

AdapthisteqAdapthisteq

Transforms pixal values using contrast-Transforms pixal values using contrast-limited adaptive histogram equalization limited adaptive histogram equalization (CLAHE)(CLAHE)

Page 13: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Image EnhancementImage Enhancement

Subdivides the image into n×m blocks, Subdivides the image into n×m blocks, calculating the histogram of each such block. calculating the histogram of each such block.

For each block, a histogram equalization is For each block, a histogram equalization is formed, which transforms the intensity values formed, which transforms the intensity values so that they are apporixmately similar. so that they are apporixmately similar.

Adapthisteq parameter “Numtiles” allows user Adapthisteq parameter “Numtiles” allows user to select n×m block.to select n×m block.

Page 14: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Image EnhancmentImage EnhancmentExample 'Numtiles', [2 2]Example 'Numtiles', [2 2]

Page 15: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Measuring AnnuliMeasuring Annuli

ManuallyManually– Allows user to click on Allows user to click on

each annuli.each annuli.

– Keeps count.Keeps count.

– Measures annuli Measures annuli distance.distance.

Semi-automaticSemi-automatic– User selects area of User selects area of

otolith to count.otolith to count.

– Keeps count.Keeps count.

– Measures annuli Measures annuli distance.distance.

Page 16: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Measuring ManuallyMeasuring Manually

Impixel lets user select any point on the Impixel lets user select any point on the image by clicking mouse.image by clicking mouse.

Outputs [x, y, intensity].Outputs [x, y, intensity].

Count the number of annuli and measure Count the number of annuli and measure distance from each point to focus of otolith.distance from each point to focus of otolith.

Page 17: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Measuring ManuallyMeasuring Manually

[77, 382, 24]

[325, 292, 33]

[103, 369, 157]

[146, 353, 118][217, 329, 62]

Distance = 113

Page 18: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Semi-automatic MeasuringSemi-automatic Measuring

ImprofileImprofile

– Computes the intensity values along a line or multiline path in Computes the intensity values along a line or multiline path in an image. an image.

Page 19: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Measuring AnnuliMeasuring Annuli

Profiles of images before and after Profiles of images before and after enhancement.enhancement.

Large Mouth Bass Age 3“ No adjustment ”

Large Mouth Bass Age 3 “ 'Numtiles', [20 20] ”

Page 20: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Counting AnnuliCounting Annuli

Polyfit Polyfit p = polyfit(x,y,n) p = polyfit(x,y,n) – Finds the coeffecients of a polynomial p(x) of Finds the coeffecients of a polynomial p(x) of

degree n that fits the data.degree n that fits the data.

– LinearLinear p = polyfit(x,y,1)p = polyfit(x,y,1)

– QuadracticQuadractic p = polyfit(x,y,2)p = polyfit(x,y,2)

Page 21: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

ExamplesExamples LinearLinear QuadraticQuadratic

Large Mouth Bass Age 3 Large Mouth Bass Age 3

Page 22: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

CountingCounting Calculate area under polyfit lineCalculate area under polyfit line

For better polyfit disregard first area For better polyfit disregard first area plotted by focus plotted by focus

Page 23: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

CountingCounting Measure residuals below the polyfit.Measure residuals below the polyfit.

Take average residual and multiply it by a stringency factor between Take average residual and multiply it by a stringency factor between 0.1 and 0.5.0.1 and 0.5.

1845 859 42010

Median = 640Stringency = 0.3Anything less that 192 will not be counted as an annlus.

Page 24: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Semi-automated AgingSemi-automated Aging

Select an area of interest.Select an area of interest.

[406,279][448,270]

[387,189]

Page 25: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Measuring AnnuliMeasuring Annuli

Takes Width at edge of area selection to Takes Width at edge of area selection to calculate number of rays.calculate number of rays.

Page 26: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

BackcalculationsBackcalculations

Growth backcalculations are one of the most Growth backcalculations are one of the most powerful applications of the otolith and are used powerful applications of the otolith and are used to estimate fish length at a previous age.to estimate fish length at a previous age. Backcalculations are a relationship between Backcalculations are a relationship between

otoliths and fish length.otoliths and fish length. Three ModelsThree Models

– Frasier-LeeFrasier-Lee– Biological InterceptBiological Intercept– Weisburg Weisburg

Page 27: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Frasier-Lee (Regression Model)Frasier-Lee (Regression Model)

Li = BCL at annulus iLi = BCL at annulus i

Lc = length at caputreLc = length at caputre

Si = otolith radius to annulus iSi = otolith radius to annulus i

Sc = total otolith radiusSc = total otolith radius

a = correction factor (used only when aging a = correction factor (used only when aging with scales otherwise = 0)with scales otherwise = 0)

Li = a + (Lc – a)(Si /Sc)

Page 28: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Biological Intercept ModelBiological Intercept Model

Modified version of the Frasier-Lee model.Modified version of the Frasier-Lee model.

Accounts for systematic variation in fish length. Otoliths of Accounts for systematic variation in fish length. Otoliths of slow-growing fish tend to be larger and heavier than fast-slow-growing fish tend to be larger and heavier than fast-growing fish of the same size.growing fish of the same size.

Biological Intercept can be determined by simple Biological Intercept can be determined by simple measurements of fish and otolith size in newly-hatched larvae measurements of fish and otolith size in newly-hatched larvae

in the laboratoryin the laboratory

Page 29: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Biological Intercept ModelBiological Intercept Model

Li = BCL at annulus iLi = BCL at annulus i

Lc = lenth at captureLc = lenth at capture

Si = otolith radius to annulus iSi = otolith radius to annulus i

Sc = total otolith radiusSc = total otolith radius

L0 = correction factor for body lengthL0 = correction factor for body length

S0 = correction factor for otolith length S0 = correction factor for otolith length

Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0)

Page 30: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Weisberg ModelWeisberg Model

Uses a linear model to separate age- and year-specific effects on otolith Uses a linear model to separate age- and year-specific effects on otolith growth.growth.

Similar to that of a two-way analysis of variance, where age of fish and Similar to that of a two-way analysis of variance, where age of fish and growth year are factors assigned to the response variable of average growth year are factors assigned to the response variable of average increment width in a growth year for a given age of fish.increment width in a growth year for a given age of fish.

The year interaction describes how environmental conditions affect fish of The year interaction describes how environmental conditions affect fish of different ages. different ages.

Expected annual growth increment = Age effect + Year effect x Year Interaction

Age1 + Year1993 = 1.7 Age2 + Year1994 = 1.4 Age3 + Year1995 = 0.9 Age1 + Year1994 = 1.9 Age2 + Year1995 = 1.2 Age3 + Year1996 = 0.7

0.70.719961996

0.90.91.21.219951995

1.41.41.91.919941994

1.71.719931993

Age3Age3Age2Age2Age1Age1YearYear

Page 31: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

Graphical User InterfaceGraphical User Interface

Page 32: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

ReferencesReferencesBranzer JC, Campana SE, Tanner DK (2004) Habitat Fingerprints for Lake Superior Coastal Wetlands Derived from

Elemental Analysis of Yellow Perch Otoliths. Transactions of the American Fisheries Society 133:692–

704

Campana, S.E. (1999) Chemistry and Composition of Fish Otoliths: pathways, mechanisms and applications

REVIEW. Mar. Ecol. Prog. Ser.188:263-297

Campana SE, Gagne JA, McLaren JW (1995) Elemental Fingerprinting of Fish Otoliths Using ID-ICPMS. Mar. Ecol.

Prog. Ser. 122:115-120

Dwyer KS, Walsh SJ, and Campana SE (2003) Age Determination, Validation and Growth of Grand Bank Yellowtail

Flounder (Limanda ferruginea). ICES Journal of MarineScience. 60:1123–1138

Edmonds JS, Moran MJ, Caputi N, Morita M (1989) Trace Element Analysis of Fish Sagittae as an Aid to Stock

Identification: Pink Snapper (Chrysophrys auratus) in Western Australia Waters. Can. J. Fish. Aquat. Sci.

46:50-54

Fablet R, Le Josse N, Benzinou, A (2004) Automated Fish Age Estimation From Otolith Images Using Statistical

Learning. ICPR 4:503-506

Francis C, Campana SE (2004) Inferring Age From Otolith Measurements: A Review and a New Approach. Can. J.

Fish. Aquat. Sci. 61: 1269-1284

Klumb RA, Bozek MA, Frie RV (2001)Validation of Three Back-calculation Models by Using Multiple

Oxytetracycline Marks Formed in the Otoliths and Scales of Bluegill × Green Sunfish Hybrids. Can. J.

Fish. Aquat. Sci. 58: 352–364

Page 33: Otolith Aging and Analysiscbs/projects/2005_presentation_stewart_william.pdfS0 = correction factor for otolith length Li = Lc + (Lc – L0)(Si – Sc)/(Sc – S0) Weisberg Model Uses

ReferencesReferences

Patterson WP, Simth GR, Lohmann KC (1993) Contintental Paleothermometry and Seasonality Using the Isotopic Composition of Patterson WP, Simth GR, Lohmann KC (1993) Contintental Paleothermometry and Seasonality Using the Isotopic Composition of

Aragonitic Otoliths of Aragonitic Otoliths of Freshwater Fishes. Geophys. Mono. 78:191-202Freshwater Fishes. Geophys. Mono. 78:191-202

Secor DH (1992) Application of Otolith Microchemistry Analysis to Investigate Anadromy in Chesapeake Bay striped bass Secor DH (1992) Application of Otolith Microchemistry Analysis to Investigate Anadromy in Chesapeake Bay striped bass Morone Morone

saxatilis. Fish. Bull., U.S. 90:798-806 saxatilis. Fish. Bull., U.S. 90:798-806

Thorrold SR, Campana SE, Jones CM, Swart PK (1997) Factors Determining ?Thorrold SR, Campana SE, Jones CM, Swart PK (1997) Factors Determining ?1313C and ?C and ?1818O Fractionation in Aragonitic Otoliths O Fractionation in Aragonitic Otoliths

of Marine Fish. Geochimica et Cosmochimica Acta. 61:2909-2919of Marine Fish. Geochimica et Cosmochimica Acta. 61:2909-2919

Tresher RE, Proctor Ch, Gunn JS, Harrowfield IR (1994) An Evaluation of Electron Probe Microanalysis of Otoliths for Stock Tresher RE, Proctor Ch, Gunn JS, Harrowfield IR (1994) An Evaluation of Electron Probe Microanalysis of Otoliths for Stock

Delineation and Identification of Nursery Areas in a Southern Temperate Groundfish. Delineation and Identification of Nursery Areas in a Southern Temperate Groundfish. Nemadactylus macropteruNemadactylus macropteruss

(Cheilodactylidae). Fish. Bull. 92:817-840(Cheilodactylidae). Fish. Bull. 92:817-840

Mathworks website:Mathworks website: http://www.mathworks.comhttp://www.mathworks.com

Otolith Research LaboratoryOtolith Research Laboratory

Bedford Institute of Bedford Institute of OceanographyOceanography

http://www.mar.dfo-mpo.gc.ca/science/mfd/otolith/english/home.htmhttp://www.mar.dfo-mpo.gc.ca/science/mfd/otolith/english/home.htm

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AcknowledgementsAcknowledgements

Arizona State UniversityArizona State University– Rosemary RenautRosemary Renaut

– Paul MarshPaul Marsh

Arizona Game and FishArizona Game and Fish– Kirk YoungKirk Young

– Scott BryanScott Bryan

– Marianne MedingMarianne Meding

– Diana RogersDiana Rogers