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North American Journal of Fisheries Management
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Evaluation of Calcein for Estimating Abundance of Lake
Trout Alevins on a Spawning Reef
a
b
J. Ellen Marsden , Kevin P. Kelsey , Jacob W. Riley
a c
& Joanna Hatt
a d
a
Rubenstein School of Environment and Natural Resources , University of Vermont , 81
Carrigan Drive, Burlington , Vermont , 05405 , USA
b
Ed Weed Fish Culture Station , 14 Bell Hill Road, Grand Isle , Vermont , 05458 , USA
c
Stantec Consulting , 30 Park Drive, Topsham , Maine , 04086 , USA
d
Warnell School of Forestry and Natural Resources , University of Georgia , 180 East Green
Street, Athens , Georgia , 30602 , USA
Published online: 18 Mar 2014.
To cite this article: J. Ellen Marsden , Kevin P. Kelsey , Jacob W. Riley & Joanna Hatt (2014) Evaluation of Calcein for
Estimating Abundance of Lake Trout Alevins on a Spawning Reef, North American Journal of Fisheries Management, 34:2,
270-275
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North American Journal of Fisheries Management 34:270–275, 2014
C American Fisheries Society 2014
ISSN: 0275-5947 print / 1548-8675 online
DOI: 10.1080/02755947.2013.869282
ARTICLE
Evaluation of Calcein for Estimating Abundance
of Lake Trout Alevins on a Spawning Reef
J. Ellen Marsden*
Rubenstein School of Environment and Natural Resources, University of Vermont, 81 Carrigan Drive,
Burlington, Vermont 05405, USA
Kevin P. Kelsey
Ed Weed Fish Culture Station, 14 Bell Hill Road, Grand Isle, Vermont 05458, USA
Downloaded by [J. Ellen Marsden] at 13:27 18 March 2014
Jacob W. Riley1 and Joanna Hatt2
Rubenstein School of Environment and Natural Resources, University of Vermont, 81 Carrigan Drive,
Burlington, Vermont 05405, USA
Abstract
Reproduction by stocked Lake Trout Salvelinus namaycush is generally estimated as the relative abundance of
fry, that is, catch per unit effort in emergent fry traps and in beam trawls, but these estimates have high variance
due to spatially heterogeneous distributions of fry. We used calcein, which produces a fluorescent mark in calcified
structures, to batch-mark fry and generate a mark–recapture estimate of fry abundance on a small, shallow spawning
reef. Eggs collected from feral Lake Trout in Lake Champlain, Vermont were reared at ambient lake temperatures,
and fry were marked 7 d after hatching. Fry were immersed in a salt solution for osmotic induction and then placed
for 4 min in a calcein solution. After marking, 18,000 fry were released on a spawning reef, and 2,000 fry were
maintained in the hatchery. Wild-caught fry and hatchery fry were checked for marks every 2–9 d. Mark clarity was
highest in the mandible and tail rays. Marks may have faded, but they did not disappear: marks were visible in the
mandible in 100% of hatchery fry after 68 d. An average of 37% of wild-caught fry had marks, yielding a Chapman
population estimate ( ± SD) of 47,486 ± 2,301. The mark–recapture estimate was within the range of fry abundance
estimated over 6 years based on egg density data and estimates of hatching success but was substantially higher than
estimated for the same year-class. This work supports prior estimates of fry abundance and provides a potential
method for assessing fry abundance on deep reefs and the success of fry stocking.
Population abundance estimates of fishes are largely based
on catch-per-unit-effort (CPUE) tomeasure relative abundance
over time or space. Actual population estimates are rare except
when mark–recapture studies are possible, or when bodies of
water can be fully sampled (such as with toxicants or draining).
Lake Trout Salvelinus namaycush early life stage sampling is
one such case: eggs can be sampled quantitatively to achieve
estimates of egg density (Perkins and Krueger 1994a, 1994b),
but fry sampling with emergent fry traps and with beam trawling for postemergent fry yield only relative CPUE data (e.g.,
Bronte et al. 1995; Marsden et al. 2005). An understanding of
the relationship between CPUE and actual fry estimates would
be valuable for evaluating mortality during egg incubation, comparing fry densities among different sites and lakes, estimating
*Corresponding author: [email protected]
1
Present address: Stantec Consulting, 30 Park Drive, Topsham, Maine 04086, USA.
2
Present address: Warnell School of Forestry and Natural Resources, University of Georgia, 180 East Green Street, Athens, Georgia 30602,
USA.
Received June 4, 2013; accepted November 20, 2013
270
Downloaded by [J. Ellen Marsden] at 13:27 18 March 2014
MARK–RECAPTURE OF LAKE TROUT FRY
fry densities at sites that are too deep for fry traps (Janssen et al.
2007), and evaluating new sampling methods (e.g., Riley et al.
2011). Mark–recapture methods may provide an opportunity to
convert CPUE into a more meaningful measure of population
abundance.
Mark–recapture of newly hatched larval fishes requires using
a noninvasive mark that will not cause high mortality. Ideally,
the mark could be batch-applied by immersion, and the mark
could be read externally without killing the fish, which would
reduce processing time and costs. Calcein as a chemical fish
mark offers two distinct advantages over other chemicals such
as oxytetracycline (Brooks et al. 1994). Immersion time in the
chemical is short (minutes rather than hours), which minimizes
stress, and the mark is visible both externally (scales and fin rays)
and internally (bones and otoliths) under fluorescent light, so
that fish do not need to be killed for mark detection. The purposes
of this study were to evaluate calcein as a mark for newly hatched
Lake Trout fry and to use calcein in a mark–recapture study of
fry emerging on a reef that has been historically sampled with
emergent fry traps (Ellrott and Marsden 2005; Marsden et al.
2005; Riley et al. 2009).
METHODS
Eggs were collected from Lake Trout adults stocked as yearlings in Lake Champlain and were reared at ambient lake temperatures at the Ed Weed Fish Culture Station to time the emergence of hatchery fry to coincide with hatching of eggs in the
lake. On March 29, 2007, five batches of approximately 4,000
newly hatched fry each (7 d posthatch) were marked. Osmotic
induction (Mohler 2003) was used to enhance fry uptake of calcein. Each batch of fry was placed in a fine-mesh open bag and
immersed for 3.5 min in an 18-L bucket containing a 50 g/L
solution of NaCl to establish an osmotic potential. The bag of
fry was then placed briefly on adsorbent paper to remove excess salt solution and transferred to a 5 g/L solution of calcein
(SE-MARK; Western Chemical, Inc., Ferndale, Washington) for
4–5 min. In each solution, fry in the bag were gently agitated to
ensure complete exposure to the NaCl or calcein, taking care to
avoid damage to the fry. The bag was transferred to a container
of freshwater to rinse off excess calcein, after which the fry were
returned to a rearing tank. Ten to 20 fry were removed from each
batch for immediate examination of the mark. The rearing tank
was uncovered, exposing it to indirect sunlight and to indirect
fluorescent light inside the hatchery building.
On April 3, approximately 18,000 of the marked fry were
placed into four plastic bags, each containing approximately
16 L of water and supplemental oxygen. The bags were taken
immediately to a spawning site less than 1 km from the hatchery;
this site, consisting of the cobble base of an emergent breakwall, has been intensively sampled for eggs and fry since 2000
(Ellrott and Marsden 2004; Marsden et al. 2005). A diver opened
each bag underwater, adjacent to the substrate, at a depth of 3–
4 m, and swam over the entire area of spawning substrate while
271
allowing fry to disperse. Fry were observed to move rapidly
downward into the substrate. A fifth bag, containing approximately 2,000 fry, was transported to the lake with the other bags
but then was returned to the hatchery to assess mortality due to
handling and for periodic examination of mark retention during
development.
After the fry were released, 10 emergent fry traps (Marsden
et al. 1988, 2005) were deployed over the area of substrate
where the fry had been released. The traps are rigid metal mesh
pyramids, 51 cm square, open at the base, with a capture bottle
at the top; an inverted funnel in the capture bottle prevents most
fry from exiting the trap. Five more traps were added on April
22 (19 d postrelease). Traps were checked for fry every 2 to
7 d until June 5 (63 d postrelease); all fry collected were frozen
immediately in a small volume of water until they could be
evaluated for the presence of a mark. Unusually high numbers
of fry (>75) in a few traps resulted in the death of a few fry.
The 2,000 marked fry that had been returned to the hatchery
were held until June 5, when all fry had completed yolk sac
adsorption and begun feeding. A small sample of fry (21–41
fry) was collected from the hatchery every 5 to 9 d, including
most of the days on which fry traps were checked; these fry
were also frozen until examination for marks.
In the laboratory, all fry were thawed and examined using
a SE-MARK detector (Western Chemical, Inc.); the detector
emits a 490-nm-wavelength light that causes calcein to reflect
yellow-green fluorescence when present. Fry were examined at
3 × magnification in low ambient light conditions to enhance
mark visibility. Marks were initially viewed by three individuals
to evaluate location and brightness categories of the marks.
Subsequently, a single individual recorded visibility of the mark
in all fry as absent, faint, or bright in the mandible, caudal
peduncle, tail rays, and fin rays.
We used a Chapman mark–recapture estimator (Chapman
1951, cited by Robson and Regier 1964) to estimate size of
the Lake Trout fry population on the spawning reef. For purposes of the estimate, only fry with a detectable mark in the
mandible or tail rays (see Results) were considered to be recaptures. Fry population estimates were compared with estimates
derived from egg collections in 2002, 2003, 2005, 2006, 2011,
and 2012 (Marsden et al. 2005; Riley et al. 2011; J. E. Marsden
unpublished data). Egg densities per square meter were extrapolated to the area of the reef (53 m2; Marsden et al. 2005) and
then multiplied by estimates of egg survival made in 2002 and
2003 (Marsden et al. 2005). Egg survival estimates ranged from
1.1% to 18.2%; for reasons explained below, we used the higher
estimate in our calculations.
RESULTS
Fry did not show signs of distress during or after marking
with calcein; mortality of marked fry in the hatchery through
emergence was less than 5% and did not differ from mortality of
unmarked fry from the same feral broodstock reared in the same
272
MARSDEN ET AL.
TABLE 1. Number and percent of hatchery Lake Trout fry (N = 262) in
which calcein marks were observed in four parts of the body. Fins = rays in
any of the paired or medial fins other than tail. Not all mark locations could be
scored for some fish due to physical damage to the specimens during thawing.
Mandible
Caudal
peduncle
Tail
rays
Fin
rays
Bright
Faint
No mark
157
97
6
29
128
105
72
149
32
4
55
200
% bright
% faint
% no mark
60.4
37.3
2.3
11.0
48.9
40.1
28.5
58.9
12.7
1.5
21.2
77.2
Downloaded by [J. Ellen Marsden] at 13:27 18 March 2014
Mark intensity
hatchery for stocking. Calcein immersion produced externally
visible marks on Lake Trout fry; the proportion of fry examined
that had a mark in a given location did not decline over the 68day observation period, indicating that the marks did not fade to
nonrecognition over that period. Marks were most readily seen
in the mandible of Lake Trout fry: 97% of fry were observed
to have a faint (37%) or bright (60%) mark on the mandible
(Table 1; Figure 1). Eighty-seven percent of fry had a mark
visible in the tail rays, 60% in the caudal peduncle, and 23%
in the fin rays. The majority of marks in locations other than
the mandible were faint (21–57%, depending on location); only
1.5–28% of fry had bright marks in these other locations. Bright
marks were unmistakable; faint marks were less obvious, and
care was taken to avoid recording reflections of the fluorescent
light sources from wet fish that resembled faint marks.
A total of 699 wild fry were collected in fry traps. Of these,
643 were examined for marks; the remaining fry were dead
when captured and were slightly decomposed, so that the tail
rays were not intact. On the basis of the hatchery fry results, and
to ensure certainty in counting marked fry, we examined only
the mandible and tail rays. Fifteen fry (2.3%) had a bright mark
in the mandible and seven (1%) had a bright mark in the tail
rays. Faint marks were observed in the mandible of 257 (40%)
fry and in the tail rays of 165 (26%) fry. If a bright mark was
seen in the tail, there was also a bright mark in the mandible;
one of the fry with a bright mark in the mandible had no mark in
the tail rays, but the remainder had bright (7) or faint (7) mark
in the tail rays.
The proportion of wild-caught fry that had a mark was reasonably consistent throughout the capture period, averaging 37%,
except on April 22 and 24 (19 and 21 d postrelease), when 88%
and 56% of fry had marks, respectively (Figure 2). The population of fry on the spawning reef estimated using all the fry
with any mark intensity in either location was 45,949 ± 2,198
( ± SD) (Figure 3). Fry population estimates from 2001 to 2012
generated from egg densities and an estimated 18.2% survival
FIGURE 1. Percent of hatchery-maintained Lake Trout fry that showed a visible calcein mark on the mandible, tail rays, caudal peduncle, and fin rays up to 68
d after marking.
Downloaded by [J. Ellen Marsden] at 13:27 18 March 2014
MARK–RECAPTURE OF LAKE TROUT FRY
273
FIGURE 2. Percentage of Lake Trout fry collected on the Grand Isle breakwall, Lake Champlain, with a faint or bright calcein mark in either the mandible or
tail rays, 15–68 d after marking and release on April 3.
FIGURE 3. Number of Lake Trout fry on the Grand Isle breakwall, Lake Champlain, estimated from egg collections in 2001–2012 and mark–recapture of fry in
2006. Error bars show standard deviations.
274
MARSDEN ET AL.
Downloaded by [J. Ellen Marsden] at 13:27 18 March 2014
to hatch ranged from 18,336 ± 12,337 to 92,823 ± 15,993 fry;
the estimate for 2007 was 18,366 ± 12,337 (Figure 3).
DISCUSSION
We used calcein marking in a novel application to estimate
population size of preemergent lake trout fry on a spawning
reef in Lake Champlain. Calcein provides a low-stress mark
procedure for very young fish, easy to apply and easy to read.
Marking success was high, and mark reading was straightforward. Although we did not need to keep marked fish alive, marks
could certainly have been read without killing the fry. Overall,
marks were seen most often in the mandible and least often in the
fin rays. The high proportion of faint marks we saw is probably
a consequence of fry not all having equal or sufficient exposure
to the calcein solution. This problem should be readily resolved
by marking smaller batches of fry, or by using a broad, shallow
tray for immersion, for greater individual exposure. Fading may
also have been a consequence of freezing and storing the fry;
although we were generally advised that this process would not
affect the marks, the effect of freezing should be quantitatively
evaluated.
The Chapman estimate of fry abundance on the spawning
reef in 2007 fell within the range of annual estimates from 2001
to 2012; it was, however, more than twice as high as the estimate generated for the 2007 year-class from egg collections in
2006 (Figure 3). The assumptions of Chapman mark–recapture
estimation were largely met. Mortality due to starvation does
not occur while fry are feeding from their yolk sac. The population of fry on the reef is closed (no emigration, immigration,
or births): fry reside within the spawning reef until emergence.
Loss due to predation probably occurred, but it is reasonable
to assume predation was equal among marked and unmarked
fry, as was found for Atlantic salmon by Mohler et al. (2002).
Marked fry were distributed widely around the reef to maximize
mixing with unmarked fry. The ratio of marked to unmarked fry
remained fairly constant during the 7 weeks of fry trapping,
except on April 22 and 24, when the ratio was higher. If marked
and unmarked fry had different susceptibility to capture, as a
consequence of disorientation or other altered behavior due to
handling and release, we would expect fry to have acclimatized
to the reef by the time traps were first checked over 2 weeks later.
Marked fry in the hatchery did not exhibit unusual mortality.
Recalculating the Chapman estimate based only on collections
after May 1 (28 d postrelease), when the proportion of marked
fry had stabilized, yielded an estimate of 48,891 ± 2,440, only
6% larger than the original estimate.
The degree to which the remaining assumption, that marks
were not lost, was met is not entirely certain. Calcein has the
disadvantage that the marks may fade in direct and artificial
sunlight (Bashey 2004; Honeyfield et al. 2008; Elle et al. 2010;
Hill and Quesada 2010). Mark loss in these studies primarily
occurred in fish held in shallow water, such as hatchery tanks and
raceways. We did not see a reduction in the proportion of marked
fry in the hatchery over the 68 d of the study; however, fry were
exposed only to filtered sunlight and dim fluorescent light. Lake
trout fry in the wild are unlikely to be exposed to direct sunlight
for long periods, as lake trout are photophobic until ages 4 to
6 weeks, remaining in dark, interstitial spaces during daylight
hours (Baird and Krueger 2000). Soon after emergence they
descend to deeper water, where sunlight is filtered, and continue
to occupy deeper water into the fall (Bronte et al. 1995). Loss
of mark intensity is also related to growth, as the marks become
more diffuse with increasing size of marked bones and fin rays
(Negus and Tureson 2004). Negus and Tureson did not see mark
intensity fade until after 12 months, when rainbow trout had
reached smolt size; because we examined fry within 9 weeks
of marking, mark intensity was unlikely to have faded due to
growth hin that period. Nevertheless, we did observe a larger
proportion of fry with faint marks relative to bright marks in
the wild-caught fry than in hatchery fry: the proportion of total
fry caught with bright marks each day declined steadily from
40% (mandible) and 33% (tail rays) to zero by May 21. These
data suggest that marks may have faded over the period of fry
emergence. However, the proportion of total fry captured that
had a mark did not change after May 8, suggesting that marks did
not disappear. If marks faded to the point that they could not be
recognized, our data would have overestimated fry abundance.
Estimates of fry abundance from egg density data could also
be flawed. Density of eggs in individual egg bags is highly variable (Riley et al. 2011), as seen particularly in 2002 (Figure 3).
Marsden et al. (2005) estimated egg survival by trapping eggs
in egg bags that were covered after spawning and then counting
the hatched fry in spring. This method yielded egg survival estimates from Grand Isle, Lake Champlain, of 1.1% and 18.2% in
2001–02 and 2002–03, respectively, and 1.8% at Whallon Bay
in Lake Champlain. The lowest of these estimates is probably
biased by the collection methods. Egg bags prevent eggs from
settling deeper than the depth of the bag (approximately 0.3 m),
so they may be vulnerable to loss or damage from turbulence.
Additionally, some bags at Grand Isle contained so many eggs
that close proximity probably caused fungus-related mortality
(Ellrott and Marsden 2004). For fry densities in 2007 to be
equivalent to our mark–recapture population estimate, hatching
success would need to approach 50%. However, this may not
be unrealistic. Once eggs are entrained into interstices, the mortality from predation by interstitial species—sculpins (Cottus
spp.) and crayfish (Orconectes spp.)—and other sources further reduce hatching success. Densities of both species on Lake
Trout spawning reefs in Lake Champlain are low compared
with sites in the Great Lakes (Marsden et al. 2005). Perkins and
Krueger (1994b) determined that egg loss due to mortality and
dislodgment overwinter in uncovered egg bags was 1.5–5.4%,
compared with 8.4–15.6% in covered bags and 14.7–39% in
incubators. Egg seeding experiments in egg bags showed that
egg retention is higher at Grand Isle than at sites in Lake Michigan (Claramunt et al. 2005; Fitzsimons et al. 2007). Modeling
of overwinter egg mortality under a range of predator density
Downloaded by [J. Ellen Marsden] at 13:27 18 March 2014
MARK–RECAPTURE OF LAKE TROUT FRY
scenarios suggests that 50% survival is highly probable, given
the predator densities at the Grand Isle site (J. W. Riley, unpublished data). The fact that the mark–recapture estimates were
within the range of independent estimates of the fry populations
at this site, extrapolated from egg densities, lends support to the
probability that this estimate is robust.
In summary, this study confirms the usefulness of calcein
for batch-marking very early stages of salmonid fry and its facility for readily observing marks in live or dead fry. Marking
effectiveness can be improved by ensuring better exposure of
all fry to the dye bath. Future studies should examine the extent to which marks may have faded in the wild. Despite these
limitations, our estimates of fry population confirm that prior
estimates derived from egg density data and estimates of survival to hatch are robust. Mark–recapture estimates would be
of particular value for assessing fry production on deep reefs,
such as the Mid-Lake Reef Complex in Lake Michigan, where
fry collection is limited to sampling by remotely operated vehicles, and density estimates are infeasible (Janssen et al. 2007;
Riley et al. 2011). Calcein would also be valuable for assessing
the strategy of stocking Lake Trout at the early fry stage, by
enabling assessment of survival within at least the first year of
life.
ACKNOWLEDGMENTS
We thank F. Steven Elle and Jerre Mohler for their assistance
and advice on use of calcein, Thomas A. Bell for loaning a
calcein detector, and David Erdahl at the U.S. Fish and Wildlife
Service W. S. Bozeman National Investigational New Animal
Drug (INAD) Office for his assistance with the INAD permit to
use calcein.
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