7190

• UCREFRP Catalog Number: 7190
• Author: Muth, R. T., T. P. Nesler and A. F. Wasowicz.
• Title: Marking Cyprinid Larvae with Tetracycline.
• USFW Year: 1988.
• USFW - Doc Type: 89-95.
• Copyright Material: YES

90
MUTH ET AL.
tore or light) to alter growth patterns on bony
structures. Many marking studies offish early-life
stages have been primarily concerned with age or
growth determinations. Few successful methods
for marking fish embryos or early larvae, specifi-
cally for application in field investigation, have
been described (e.g., Muncy and D'Silva 1981;
Brothers 1985; Tsukamoto 1985). Brothers (1985)
and Tsukamoto (1985) considered otoliths ideal
for marking because they are the first permanent
calcified structures present in the earliest life
history stages of fish and are, effectively, biolog-
ical internal tags. We concluded that tetracycline
marking of otoliths would most likely meet our
requirements for an optimal mark. Tetracycline
compounds are well-known markers for calcified
structures, and they fluoresce yellow under ultra-
violet (UV) light (Milch et al. 1957; Weber and
Ridgway 1962, 1967; Choate 1964). They have
been used to mark larvae of several fish species,
for example, pinfish Lagodon rhomboides and
spot Leiostomus xanthurus (Hettler 1984) and ayu
Plecoglossus altivelis (Tsukamoto 1985), but their
application to cypriniform larvae has not been
reported.
Methods
Approximately 1,500 4-d-old (posthatching)
Colorado squawfish protolarvae (larvae prior to
development of median fin rays, sensu Snyder
1981) were obtained from the Dexter (New Mex-
ico) National Fish Hatchery in June 1986. The
larvae were placed in aflow-through holding
trough that received 20°C well water; 20-22°C
water was used by Hamman (1986) for hatchery
culture of Colorado squawfish embryos and lar-
vae. Most larvae were swim-up, still had 50-70%
of their yolk, and measured 7.5-8.0 mm total
length (TL). The sagitta and lapillus otoliths were
both present. After a 24-h acclimation period,
dead, damaged, or atypically behaving larvae
were removed. The remaining larvae were divided
into ten 140-specimen experimental groups.
Nine experimental groups were treated by plac-
ing the larvae in 1-L glass beakers containing
tetracycline hydrochloride (TC) solutions of 200,
350, or 500 mg TC/L aerated distilled water.
Distilled water was used as the diluent because
TC binds to calcium, and hard water, which
contains calcium ions, might hinder the uptake of
TC by the otoliths. Each TC test solution was
adjusted to pH 6.8-7.0 with tris buffer (Mettler
1984). The pH prior to buffering was about 3.6.
Exposure times for each concentration were 4, 12,
and 36 h. During their exposure, the treatment
groups were aerated and incubated in a covered
water bath at 20°C, but they were not fed. The
10th experimental group was maintained as a
control.
After each prescribed exposure, the treated
larvae were removed from the test solutions, and
the numbers of dead larvae were recorded. Sur-
viving larvae were placed in aerated well water in
3.8-L rearing jars and incubated in a water bath at
20°C. Control larvae were taken directly from the
holding trough and placed in a rearing jar. Rearing
jars were partially shaded with sheets of opaque
plastic. The photoperiod was about 9-h light: IS-h
dark; light intensity was 1001x. Larvae were fed
twice daily with live artemia Artemis sp. nauplii
and TetraMin Fry Diet. After about 4 weeks,
TetraMin Staple Food was included in the diet
(Muth et al. 1985). Before each feeding, the rear-
ing jars were cleaned, and the numbers of dead
larvae were recorded. Larvae were reared for up
to 77 d after treatment.
Ten living larvae from each experimental group
were preserved and measured (TL) immediately
after treatment and at weekly intervals thereafter.
Two different preservation fluids were tested, and
their effects on otolith structure and on TC-pro-
duced marks were compared. At each time inter-
val, five larvae were fixed and preserved in 95%
ethanol (pH = 8.0), and five were fixed and
preserved in formalin solutions buffered to pH 6.8
with phosphate (Markle 1984). Larvae were fixed
in 10% buffered formatin for 24 h, then transferred
to 3% buffered formalin for storage. Samples were
stored in the dark until they were examined 3-4
months after treatment.
Two whole larvae from each sample lot were
examined with incident UV light under adissect-
ing microscope for the presence of external fluo-
rescent marks. Sagittae and lapilli were extracted
from larvae by procedures similar to those de-
scribed by Brothers (1987) for embryonic and
larval fish. Otoliths were mounted in glycerin on
glass slides and examined with incident UV light
under a compound microscope for the presence
and intensity of fluorescent marks. The average
time spent on the extraction and mounting of
otoliths was about 10 min/specimen. The intensity
of the mark was determined for each of the larvae
that was examined, and it was ranked by a pro-
cedure similar to that used by Tsukamoto (1985).
The ranking categories that we designated were
absent, faint, lucid, or bright, corresponding to
values of 0, 1, 2, or 3, respectively. Individual