alternative methods for marking otoliths: enriched stable isotopes and fluorescent dyes andrew r....
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![Page 1: Alternative methods for marking otoliths: enriched stable isotopes and fluorescent dyes Andrew R. Munro](https://reader038.vdocuments.net/reader038/viewer/2022110208/56649d955503460f94a7dbc7/html5/thumbnails/1.jpg)
Alternative methods for marking otoliths: enriched stable isotopes
and fluorescent dyes
Andrew R. Munro
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> 60 million native fish stocked in MDB over past 30 years
Fate of stocked fish unknownsuccess of stockingeffects on ecology
Lack of suitable methods for marking hatchery fish
CWTAlizarin complexone
Murray-Darling Basin
Native fish stocking - Australia
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Enriched stable isotopesOtolith marking experiments
Fingerling immersion Larval immersionBroodstock injection (transgenerational marking)
Osmotic induction of fish with fluorescent compounds
Developing methods for marking hatchery fish
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Stable isotopes
Barium
natural relative abundances
137Ba = 11.30% 138Ba = 71.70%
137Ba 11.30
138Ba 71.70= = 6.38
Non radioactive
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Enriched stable isotopes
0
20
40
60
80
100
Natural Enriched
% a
bu
nd
ance
137Ba
138Ba
81.90%11.30%
BaCO3 – enriched in 137Ba
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Enriched Isotopes
Hypothesis: can alter otolith isotopic ratios by exposing fish to specific isotopes
Enriched Ba added (µg/L)
0 5 10 15 20
138 B
a/13
7 Ba
0
2
4
6
8
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Fingerling immersion
Reared juvenile golden perch in varying levels of enriched Ba for different lengths of time
1d 4d 8d 24d
0 µg/L 1 µg/L 5 µg/L 15 µg/L
Munro et al. (2008) CJFAS
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Analysis
Otolith analysis: either whole or sectioned otoliths
Transects/spot analyses
Measured isotopes of interest (e.g.137Ba & 138Ba)
LA-ICPMS (single collector)
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8 d immersion – 15 µg137Ba/L
Fingerling immersion results
Distance from edge (m)
020406080100120140
13
8 Ba
/13
7 Ba
0
2
4
6
8
Ba rationatural ratio
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Length of immersion (d)
control 1 4 8 24
13
8 Ba/1
37 B
a
0
2
4
6
1 µg/L5 µg/L Ba15 µg/L Ba
Fingerling immersion results
Significant mark 1 d @ 15 µg/L
100% marked 8 d @ 15 µg/L
Base water concentration ~ 15 µg/L Munro et al. (2008) CJFAS
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Fingerling immersion summary
• Altered otolith Ba isotope ratio
• 100% mark success – 8 d @ 15µg/L
• Unambiguous mark – not natural
• Stress free
• Requires extended holding time
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0 h 1 d 21 d
20 µg/g 2 fish 2 fish X
40 µg/g 2 fish 2 fish 2 fish
Maternal dose rate (137Ba)
Length of time prior to hormone injection
Brood stock injection
Munro et al. (2009) JFB
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Distance from edge (µm)
0 10 20 30 40 50 60
138 B
a/13
7 Ba
0
2
4
6
8
Ba ratio
natural Ba ratio
Maternal parent injected with 40 µg/g of enriched 137Ba at same time hormone
Brood stock injection results
nucleus
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Conc. (µg/g)
Injection (d)
4020Treatment
Control 0 1 0 1 21
138 B
a/137 B
a
0
1
2
3
4
5
6
7
Brood stock injection results
100%
Munro et al. (2009) JFB
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• Altered otolith Ba isotope ratio
• 40 µg/g at time of hormone 100% mark
• Fits in with standard hatchery practices
• Variable spawning success of injected fish
Brood stock injection summary
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Larval immersion
Reared larval golden perch in varying levels of enriched Ba for different lengths of time
1d 2d 3d 5d
0 µg/L 30 µg/L 60 µg/L 90 µg/L
Woodcock et al. (2011) EFF
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Larval immersion results
Days exposure
Control 1 2 3 5
138 B
a/13
7 Ba
0
1
2
3
4
5
6
7
30 µg/L60 µg/L 90 µg/L
100 % mark success
Base water concentration ~ 10 µg/L Woodcock et al. (2011) EFF
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Larval immersion summary
• compatible with hatchery procedures
• 100% mark success
• high density = less isotope
• mark location known
• most cost effective
• variable survival to stocking• 30–50%
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Calcein marked golden perch otoliths
Method developed for marking Atlantic salmon with calcein (Mohler 2003)
Also trialed alizarin red S as a cheaper alternative
5% Salt Freshwater Calcein
3.5 min 5 minRinse
Osmotic induction marking
Crook et al. (2007, 2009) NAJFM
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Control Calcein treatment ARS treatment
Dissecting microscope - white light
Dissecting microscope - fluorescence filters
9 months post-marking
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Control Calcein treatment ARS treatment
Dissecting microscope - white light
Dissecting microscope - fluorescence filters
9 months post-marking
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Control Calcein treatment
Dissecting microscope - white light
Dissecting microscope - fluorescence filters
ARS treatment
9 months post-marking
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Practical and objective way of identifying
marks on live fish in the field
Non-lethal field detection
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GFP portable fluorometer
Calcein marked fish – 26 months
Billabong Ck Edward R Narrandera
Sam
ple
:ref
eren
ce f
luo
resc
ence
rat
io
Unmarked fish
Reference
0
0.5
1
1.5
2 Signal overload
Calcein marked
n=12
n=12n=32
n=20
Non-lethal field detection
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Advantages:
Marking procedure is easy and quick (15 min)
Detectable on live fish in the field
Excellent accuracy (100% after 18 months in field, 26 months in lab)
Disadvantages:
Adjustments to hatchery protocols required
Chemicals must be disposed of appropriately
Unknown longevity of external marks
Osmotic induction marking summary
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Method $ per 1,000 fingerling Notes
Isotope immersion (137Ba)
fingerlings 9.80 15 µg/L @ 10 fish/L
larvae 1.60 30 µg/L @ 250 fish/L
Isotope injection (137Ba) 0.66 - 19.14 20 µg/g
Osmotic induction
Calcein 37.00 0.5% @ 800 fish/L
ARS 0.50 0.05% @ 800 fish/L
Marking Costs
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Cost comparison for marking
Method $ per 1,000
Immersion
fingerling 9.80
larvae 1.60
Injection 0.66 - 19.14
Calcein (OI) 37.00
ARS (OI) 0.50
Thermal 6.25
CWT 83
ALC (10-400 mg/L) 11.48 - 459
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Cost comparison for reading marks
reading cost/fish
Enriched isotopes $14.50 - $45.00
Calcein & field detector $0.00
Thermal marking $5.00 - $14.00
Coded wire tags $2.14
Alizarin complexone $3.75
marking/1,000
$1.60
$37.00
$6.25
$83.00
$11.48
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Scaling up
Osmotic induction Calcein marking
• 60,000 fish• batches ~4,000 – 5,000 fish
Larval marking w/isotopes
- ~100,000 fish- few ml isotope solution
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Multiple enriched stable isotopes4 Ba isotopes 15 unique combinations
Multiple unique batch marks
138Ba/136Ba
0 10 20 30 40
138 B
a/1
37 B
a
0
5
10
15
20
25
NaturalControl136Ba137Ba138Ba136Ba & 137Ba136Ba & 138Ba137Ba & 138Ba136Ba, 137Ba & 138Ba
3 isotopes7 unique marks
Woodcock et al. (2011) EFF
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Many more if include isotopes of other elements (e.g. Sr, Mg)
Multiple unique batch marks
88Sr/86Sr
3 4 5 6 7 8
138 B
a/13
7 Ba
3
4
5
6
7
n = 6/treatment
error bars: 95% CI
0 µg/L100 µg/L 25 µg/L
0 µg/L
1 µg/L
15 µg/L
8 unique marks; 96% mark success Munro et al. (2008) CJFAS
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All methods able to produce distinctive mark in fish otoliths
Most cost effective method larval immersion in enriched isotopes
Combine with osmotic induction at fingerling stage
external & internal mark
Investigate effects on growth & survival
Investigate survival & dispersal of stocked fish, & impacts of stocking
Methods have potential for use in other areas (e.g. larval dispersal)
Summary
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Acknowledgements
Co-investigators: Bronwyn M. GillandersDavid A. CrookSkye H. WoodcockAndrew C. Sanger
Funding: Australian Research CouncilMurray-Darling Basin Authority
And many others who have helped.Photo: NSW Fisheries - The Fisherman, 1962