This study compared the effectiveness of oxytetracycline (OTC) and calcein for marking fish otoliths to aid in age validation. Juvenile pinfish were immersed in various concentrations of OTC or calcein for different time periods. Their otoliths were later examined under fluorescence microscopy. Results showed calcein produced stronger marks than OTC. Higher calcein concentrations and using a NaCl solution improved mark strength. Calcein marks remained clearly visible over several months. This technique can help scientists accurately determine the age of fish by marking otoliths at a known time.

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Brown, Michael L., Powell, Jennifer L., & Lucchesi, David O., 2002. In-transit
oxytetracycline marking, nonlethal mark detection, and tissue residue depletion in yellow
perch. North American Journal of Fisheries Management​. 22: 236-242.
Denson, Michael R. & Smith, Theodore I.J., 2008. Use of tetracycline to mark larval red
drum: survival and mark persistence. North American Journal of Fisheries
Management. 28: 1779-1789.
Fielder, David G., 2002. Methodology for immersion marking walleye fry and
fingerlings in oxytetracycline hydrochloride and its detection with fluorescence
microscopy. State of Michigan Department of Natural Resources. Fisheries Technical
Report 2002-1: 1-21.
Geffen, A.J., 1992. Validation of otolith increment deposition rate. Can. Spec. Publ. Fish.
Aquat. Sci. 117: 101-113.
Hettler, William F., 1984. Marking otoliths by immersion of marine fish larvae in tetracycline.
Transactions of the American Fisheries Society. 113:370-373.
LÜ, Hongjian, Zhang, Xiumei, Xi, Dan, & Gao, Tianxiang, 2014. Use of calcein and alzarin
red S for immersion marking of black rockfish Sebastes schlegelii juveniles.
Chinese Jouranl of Oceanography and Limnology. 32: 88-98.
McFarlane, G.A. & Beamish, R.J., 1987. Selection of dosages of oxytetracycline for age
validation studies. Can. J. Fish. Aquat. Sci. 44: 905-909.
Reinert, Thomas R., Wallin, Julie, Griffin, Mary C., Conroy, Michael J., & Van Den Avyle,
Michael J., 1997. Long-term retention and detection of oxytetracycline marks
applied to hatchery-reared larval striped bass, Morone saxatilis. Can. J. Fish. Aquat. Sci.
55:539-543.
Taylor, M.D., Fielder, D.S., & Suthers, I.M., 2004. Batch marking of otoliths and fin spines
to asses the stock enhancement of Argyrosomus japonicus. Journal of Fish Biology.
66:1149-1162.
Wartenberg, Reece, Booth, Anthony J., & Weyl, Olaf L.F., 2011. A comparison of three
techniques for fluorochrome marking of juvenile Clarias gariepinus otoliths.
African Zoology​. 46(1):72-77.
Wilson, Charles A., Beckman, Daniel W., & Dean, John Mark, 1987. Calcein as a
fluorescent marker of otoliths of larval and juvenile fish. Transaction of the American
Fisheries Society. 116: 4, 668-670.
Comparison of Fish Otolith Marking Techniques to Aid in Age
Validation
M. Pappas, K. Riley, & J. Morris
NOAA Fisheries Service Southeast Fisheries Science Center Laboratory, Pivers Island, Beaufort, NC
Materials & Methods
Collection
Juvenile pinfish, Lagodon rhomboides, (4.7 ± 0.7 cm TL; 1.6 ± 0.7 g) were
collected using baited minnow traps from floating docks at the Beaufort NOAA
laboratory and held in quarantine until treated.
Chemical Immersions
Chemical solutions of OTC were prepared at concentrations of 250mg/L and
500mg/L and solutions of calcein were prepared at concentrations of 125mg/L
and 250mg/L.
Fish were immersed in 10-gal. treatment tanks of the solution for various
immersion times (anywhere from 1.5h to 9h depending on chemical type). The
type of water used in immersion was also varied, either natural seawater or a
solution of NaCl (Hettler, 1984). Treated fish were then placed in holding tanks
under natural conditions for five weeks.
Otolith Removal and Analysis
After five weeks of growth, fish were anesthetized with MS-222 and the sagittal
pair of otoliths was pulled using a dissecting microscope. Whole otoliths were
mounted onto slides and examined under fluorescent microscopy. The
chemical mark on the otolith was assigned a qualitative mark strength (no mark
or unreadable mark, weak mark, moderate mark, and strong mark (Figure 2)).
Otoliths were also removed from fish in January to determine if time had an
effect on mark strength.
Acknowledgments
Introduction
Fisheries management relies on accurate stock
assessments, which require age curves of a population. The age of a
fish is usually determined by counting annual rings in a fish’s otolith,
the calcified structure located in the cavity behind the eye. However,
because only a few species have had their ages validated, it is only
an assumption that each ring represents a year.
This study aims to find an effective technique of marking fish
with chemicals that bind to their calcified structures to enable
scientists to determine an accurate time assessment for each ring in
an otolith. This marking chemical will then be applied to wild-caught
fish via immersion baths at a known time. Fish will grow for a given
time after treatment, and when the otolith is removed, the chemical
mark will indicate a baseline from which rings can be counted
outward. Each ring can then be assigned a time, and an accurate
age curve can be constructed. This study examines the strength of
the mark on the otolith of fish treated in two different chemicals,
oxytetracycline (OTC) and calcein, as well as multiple concentrations
and multiple immersion times.
Figure 4.
Frequency distribution of otoliths comparing mark
strength of fish treated with 125mg/L of calcein in
NaCl solution and fish treated with 125mg/L of
calcein in natural seawater. n = 40
Results & Implications
• The mark strength analysis comparing chemical types showed that calcein
was a better marking agent overall (Figure1), and that calcein resulted in
more otoliths with readable marks.
• Mark strength was determined by how clearly the marking agent dyed the
otolith (Figure 2).
• Concentration of the chemical solution affected the mark strength for fish
treated with OTC (Figure 3), showing that increasing the concentration to
500mg/L resulted in stronger marks.
• Chi-squared tests of independence show that there is a significant
difference between mark strength of fish marked with calcein in natural
seawater and fish marked with calcein in a solution of NaCl. Figure 4
shows the effectiveness of using the NaCl solution during treatment of
calcein.
• Calcein was the most effective marking agent resulting in clearly defined
marks for both otoliths pulled in the summer (Figure 2) and those pulled in
the winter (Figure5).
Figure 1​.
Frequency distribution of otoliths comparing mark strength of fish
treated with calcein and fish treated with OTC. n = 80 for calcein,
n = 196 for OTC.
Figure 2.
Otoliths removed in the summer marked with OTC and calcein of
various mark strengths. The first two pictures show otoliths marked in
OTC, the first having a strong mark and the second having an
unreadable mark. The last two pictures are otoliths marked in calcein,
showing a weak mark and a strong mark respectively.
Figure 3.
Frequency distribution of otoliths comparing mark strength of
fish treated with OTC at concentrations of 250mg/L and
500mg/L. n = 40.
Literature Cited
Figure 5.
Otoliths removed in the winter marked with OTC and calcein of
various mark strengths. OTC treated otoliths are on the left and
calcein treated otoliths are on the right.
This research was made possible by the NOAA Holling’s Scholarship Program and summer
internship provided by the NOAA Office of Education. I would like to thank my mentors and co-authors
of my project, Dr. Ken Riley and Dr. James Morris. Thank you to Troy Rezek and other laboratory staff
at the NOAA Beaufort lab for facilitating my project.
This work will be presented as my marine science thesis for my undergraduate degree at Eckerd
College. I would like to thank my advisors and thesis committee, Dr. William Szelistowski, Dr. Koty
Sharp, and Dr. David Hastings for their attention to my project and work that will continue until my
defense.
Brown, Michael L., Powell, Jennifer L., & Lucchesi, David O., 2002. In-transit
oxytetracycline marking, nonlethal mark detection, and tissue residue depletion in yellow
perch. North American Journal of Fisheries Management​. 22: 236-242.
Denson, Michael R. & Smith, Theodore I.J., 2008. Use of tetracycline to mark larval red
drum: survival and mark persistence. North American Journal of Fisheries
Management. 28: 1779-1789.
Fielder, David G., 2002. Methodology for immersion marking walleye fry and
fingerlings in oxytetracycline hydrochloride and its detection with fluorescence
microscopy. State of Michigan Department of Natural Resources. Fisheries Technical
Report 2002-1: 1-21.
Geffen, A.J., 1992. Validation of otolith increment deposition rate. Can. Spec. Publ. Fish.
Aquat. Sci. 117: 101-113.
Hettler, William F., 1984. Marking otoliths by immersion of marine fish larvae in tetracycline.
Transactions of the American Fisheries Society. 113:370-373.
LÜ, Hongjian, Zhang, Xiumei, Xi, Dan, & Gao, Tianxiang, 2014. Use of calcein and alzarin
red S for immersion marking of black rockfish Sebastes schlegelii juveniles.
Chinese Jouranl of Oceanography and Limnology. 32: 88-98.
McFarlane, G.A. & Beamish, R.J., 1987. Selection of dosages of oxytetracycline for age
validation studies. Can. J. Fish. Aquat. Sci. 44: 905-909.
Reinert, Thomas R., Wallin, Julie, Griffin, Mary C., Conroy, Michael J., & Van Den Avyle,
Michael J., 1997. Long-term retention and detection of oxytetracycline marks
applied to hatchery-reared larval striped bass, Morone saxatilis. Can. J. Fish. Aquat. Sci.
55:539-543.
Taylor, M.D., Fielder, D.S., & Suthers, I.M., 2004. Batch marking of otoliths and fin spines
to asses the stock enhancement of Argyrosomus japonicus. Journal of Fish Biology.
66:1149-1162.
Wartenberg, Reece, Booth, Anthony J., & Weyl, Olaf L.F., 2011. A comparison of three
techniques for fluorochrome marking of juvenile Clarias gariepinus otoliths.
African Zoology​. 46(1):72-77.
Wilson, Charles A., Beckman, Daniel W., & Dean, John Mark, 1987. Calcein as a
fluorescent marker of otoliths of larval and juvenile fish. Transaction of the American
Fisheries Society. 116: 4, 668-670.
Comparison of Fish Otolith Marking Techniques to Aid in Age
Validation
M. Pappas, K. Riley, & J. Morris
NOAA Fisheries Service Southeast Fisheries Science Center Laboratory, Pivers Island, Beaufort, NC
Materials & Methods
Collection
Juvenile pinfish, Lagodon rhomboides, (4.7 ± 0.7 cm TL; 1.6 ± 0.7 g) were
collected using baited minnow traps from floating docks at the Beaufort NOAA
laboratory and held in quarantine until treated.
Chemical Immersions
Chemical solutions of OTC were prepared at concentrations of 250mg/L and
500mg/L and solutions of calcein were prepared at concentrations of 125mg/L
and 250mg/L.
Fish were immersed in 10-gal. treatment tanks of the solution for various
immersion times (anywhere from 1.5h to 9h depending on chemical type). The
type of water used in immersion was also varied, either natural seawater or a
solution of NaCl (Hettler, 1984). Treated fish were then placed in holding tanks
under natural conditions for five weeks.
Otolith Removal and Analysis
After five weeks of growth, fish were anesthetized with MS-222 and the sagittal
pair of otoliths was pulled using a dissecting microscope. Whole otoliths were
mounted onto slides and examined under fluorescent microscopy. The
chemical mark on the otolith was assigned a qualitative mark strength (no mark
or unreadable mark, weak mark, moderate mark, and strong mark (Figure 2)).
Otoliths were also removed from fish in January to determine if time had an
effect on mark strength.
Acknowledgments
Introduction
Fisheries management relies on accurate stock
assessments, which require age curves of a population. The age of a
fish is usually determined by counting annual rings in a fish’s otolith,
the calcified structure located in the cavity behind the eye. However,
because only a few species have had their ages validated, it is only
an assumption that each ring represents a year.
This study aims to find an effective technique of marking fish
with chemicals that bind to their calcified structures to enable
scientists to determine an accurate time assessment for each ring in
an otolith. This marking chemical will then be applied to wild-caught
fish via immersion baths at a known time. Fish will grow for a given
time after treatment, and when the otolith is removed, the chemical
mark will indicate a baseline from which rings can be counted
outward. Each ring can then be assigned a time, and an accurate
age curve can be constructed. This study examines the strength of
the mark on the otolith of fish treated in two different chemicals,
oxytetracycline (OTC) and calcein, as well as multiple concentrations
and multiple immersion times.
Figure 4.
Frequency distribution of otoliths comparing mark
strength of fish treated with 125mg/L of calcein in
NaCl solution and fish treated with 125mg/L of
calcein in natural seawater. n = 40
Results & Implications
• The mark strength analysis comparing chemical types showed that calcein
was a better marking agent overall (Figure1), and that calcein resulted in
more otoliths with readable marks.
• Mark strength was determined by how clearly the marking agent dyed the
otolith (Figure 2).
• Concentration of the chemical solution affected the mark strength for fish
treated with OTC (Figure 3), showing that increasing the concentration to
500mg/L resulted in stronger marks.
• Chi-squared tests of independence show that there is a significant
difference between mark strength of fish marked with calcein in natural
seawater and fish marked with calcein in a solution of NaCl. Figure 4
shows the effectiveness of using the NaCl solution during treatment of
calcein.
• Calcein was the most effective marking agent resulting in clearly defined
marks for both otoliths pulled in the summer (Figure 2) and those pulled in
the winter (Figure5).
Figure 1​.
Frequency distribution of otoliths comparing mark strength of fish
treated with calcein and fish treated with OTC. n = 80 for calcein,
n = 196 for OTC.
Figure 2.
Otoliths removed in the summer marked with OTC and calcein of
various mark strengths. The first two pictures show otoliths marked in
OTC, the first having a strong mark and the second having an
unreadable mark. The last two pictures are otoliths marked in calcein,
showing a weak mark and a strong mark respectively.
Figure 3.
Frequency distribution of otoliths comparing mark strength of
fish treated with OTC at concentrations of 250mg/L and
500mg/L. n = 40.
Literature Cited
Figure 5.
Otoliths removed in the winter marked with OTC and calcein of
various mark strengths. OTC treated otoliths are on the left and
calcein treated otoliths are on the right.
This research was made possible by the NOAA Holling’s Scholarship Program and summer
internship provided by the NOAA Office of Education. I would like to thank my mentors and co-authors
of my project, Dr. Ken Riley and Dr. James Morris. Thank you to Troy Rezek and other laboratory staff
at the NOAA Beaufort lab for facilitating my project.
This work will be presented as my marine science thesis for my undergraduate degree at Eckerd
College. I would like to thank my advisors and thesis committee, Dr. William Szelistowski, Dr. Koty
Sharp, and Dr. David Hastings for their attention to my project and work that will continue until my
defense.
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