Fey et al.: Daily deposition of growth increments in sagittae and lapilli of larval Esox lucius 
303 
the age validation of larval and juvenile fish (for a re¬ 
view, see Geffen, 1992; Campana, 2001). The most used 
method involves analysis of larvae of known age (e.g., 
Folkvord et al., 1997; Folkvord et al., 2000; Hill and 
Bestgen, 2014; Ding et al., 2015) and marking otoliths 
with alizarin or tetracycline (Secor et al., 1995; Fox et 
al., 2003). The marginal increment analysis method is 
more commonly used for adult fish, but it can be ap¬ 
plied to larvae (Sepulveda, 1994; Fey, 2002). Although 
most of such otolith microstructure analysis is per¬ 
formed with light microscopes, scanning electron mi¬ 
croscopes can be used as well, especially if increment 
deposition occurs at low temperatures (Radtke and Fey, 
1996) or during periods of starvation (Jones and Broth¬ 
ers, 1987; Fox et al., 2003). 
The present study validates the aging method for 
larval northern pike (Esox lucius), a species of signifi¬ 
cant importance for both commercial and recreational 
fisheries and having a wide circumpolar distribution 
(i.e., in North America and Europe) (Craig, 2008). Al¬ 
though many populations of northern pike are not over- 
exploited, in some geographical areas the species is 
close to extirpation, which is the case in many coastal 
areas of the Baltic Sea (Nilsson et al., 2014; Larsson et 
al., 2015; Skov and Nilsson, 2018). The reasons for pop¬ 
ulation declines are over-exploitation, disappearance of 
suitable spawning grounds, and low recruitment (Lars¬ 
son et al., 2015; Skov and Nilsson, 2018). Other fac¬ 
tors related to human activities in coastal areas (e.g., 
presence of wind farms and underwater cables) should 
be considered, as well (Fey et al., 2019). Given their 
endangered status in some regions, there is a need to 
better understand the early life period, growth, and 
survival of northern pike. Therefore, it is also impor¬ 
tant to evaluate the methods applied for aging larvae 
and early juveniles (e.g., by enumeration of daily incre¬ 
ments within otoliths). Unfortunately, only one publi¬ 
cation validating age estimates of northern pike from 
lapilli is available (Wang and Eckmann, 1992), and no 
information of this kind exists on the basis of sagittae. 
Although Wang and Eckmann (1992) provided general 
information on sagittae and stated that lapilli were al¬ 
ways much clearer to read than sagittae, no data have 
been presented to date on the rate of increment deposi¬ 
tion for this otolith type. 
Our goal was to estimate the periodicity of incre¬ 
ment formation and the time of the first increment 
deposition on sagittae and lapilli of known-age labora¬ 
tory-reared larval northern pike (9-33 mm in standard 
length [SL]). Changes in otolith size are described in 
relation to larval age, SL, and weight. 
Material and methods 
Rearing of fish larvae 
Eyed-eggs of northern pike (12 days after fertilization) 
from wild spawners were transported in April 2015 
from the Komorowo Hatchery (northern Poland) to the 
laboratory of the Center of Aquaculture and Ecological 
Engineering, University of Warmia and Mazury in 01- 
sztyn. For the purpose of forcing the mass hatching of 
larvae, the eggs were subjected to a temperature that 
increased within one hour by 3-4°C. Pike larvae were 
kept in two separate aquaria with a water volume of 
60 L each and initially at a density of approximately 
22 indivduals/L per aquarium. Water in the aquaria 
was aerated and purified with a bio-filter at a con¬ 
stant water temperature of 18°C (standard deviation 
[SD] 0.5°C). The larvae were fed ad libitum every 1.5 
h during day light hours with Perla Larva Proactive 
4.0 1 commercial starter feed (Skretting, Stavanger, 
Norway). The aquaria were cleaned every morning, 
before feeding began, and dead larvae were removed. 
Throughout the rearing period, a natural photoperiod 
(14 h light/10 h dark) was applied that was character¬ 
istic of the spring photoperiod in East Central Europe. 
Otolith analysis 
A sample of 25 specimens was collected from each tank 
7, 14, 22, and 28 days after hatching (dah)]. Additional¬ 
ly, 15 specimens were collected on the day of hatching. 
The fish samples were collected between 10:00 h. and 
11:00 h. The fish were preserved in 96% alcohol. Oto¬ 
liths (left and right sagittae and lapilli) were extracted 
from each larva and placed on microscope slides, dis¬ 
tal surface down (sulcus up) and covered with DEPEX 
mounting medium (Electron Microscopy Sciences, Hat¬ 
field, PA). The otoliths without any preparation were 
used in the following analysis. At the time of otolith 
extraction, the SL of each larva was measured to the 
nearest 0.1 mm, and wet weight was estimated to the 
nearest 0.01 g. Length measurements were corrected 
for shrinkage before further analysis (Greszkiewicz and 
Fey, 2018). The length (i.e., maximum diameter) of all 
extracted otoliths (n =230 pairs) was measured by us¬ 
ing an image analysis system (Image-Pro Premier, Me¬ 
dia Cybernetics, Inc., Rockville, MD) under an Eclipse 
80i transmitted light microscope (Nikon Corp., Tokyo, 
Japan). The mean value of the length measurements 
from the left and right otoliths was used for our analy¬ 
sis. The total number of increments was counted by 
the same person on two different occasions. If the dif¬ 
ference in estimated numbers exceeded a given number 
of increments (2 increments for 7 dah, 3 for 14 dah and 
22 dah, and 4 for 28 dah ), the otolith was excluded 
(approximately 14-29% of the otoliths examined). Oth¬ 
erwise, the mean value from the two readings was used 
for the analysis. Some otoliths were excluded without 
increment counts if there was a lack of confidence in 
recognizing increments. The otoliths extracted on the 
day of hatching of the larvae were used to confirm the 
position of the hatching check on otoliths of older fish. 
1 Mention of trade names or commercial companies is for iden¬ 
tification purposes only and does not imply endorsement by 
the National Marine Fisheries Service, NOAA. 
