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INTRODUCTION
While many studies have examined pectoral fin movement and
function in adult fish, less attention has been given to pectoral fins
at the larval stage of development. For juveniles and adults of many
fish species, pectoral fins act as primary propulsors during rhythmic
swimming (e.g. Webb, 1973; Blake, 1979; Drucker and Jensen,
1996a; Drucker and Jensen, 1996b; Walker and Westneat, 1997;
Hale et al., 2006), and in arrhythmic movements such as braking
(e.g. Drucker and Lauder, 2003; Higham et al., 2005) and
maneuvering (e.g. Drucker and Lauder, 2001; Drucker and Lauder,
2003). For larval fish, the pectoral fins move actively during
rhythmic swimming (Batty, 1981; Müller and van Leeuwen, 2004;
Thorsen et al., 2004), routine turning (Danos and Lauder, 2007) and
feeding (Budick and O’Malley, 2000) behaviors, yet striking
differences from adults in size and other aspects of morphology
may result in different functional demands on the pectoral fins.
Much of the work examining the behavior of fish at early
developmental stages has been on the larval zebrafish, a genetic
model system that has been used broadly to examine motor control
and movement (e.g. Fuiman and Webb, 1988; Müller et al., 2000;
Thorsen et al., 2004; Danos and Lauder, 2007; McLean et al.,
2007). Larval zebrafish beat their pectoral fins, alternating them
rhythmically in combination with body undulation during
swimming at low speed [~1–6 total body lengths (TL)s–1]. This
distinct movement pattern has been broadly referred to as ‘slow
swimming’ or ‘slow start’ (Budick and O’Malley, 2000; Müller
and van Leeuwen, 2004; Thorsen et al., 2004). During faster
swimming, the body undulates but the pectoral fins remain
positioned close to the body. While the basic patterns of
undulatory movement and the coordination of body undulations
with the pectoral fins have been described, variation of pectoral
fin and body kinematics through the duration of the swim bout
and with speed have not been examined in larvae. More broadly,
the potential locomotor functions of rhythmic pectoral fin
movements during forward swimming have not been been tested.
The first goal of this work was to measure basic kinematic
variables of the slow swim gait and to determine how these
kinematic variables change with swimming speed. Studies on adult
and juvenile pectoral fin swimmers have demonstrated that these
animals increase swimming speed by increasing fin beat frequency
and amplitude (e.g. Gibb et al., 1994; Mussi et al., 2002; Hale et
al., 2006), in some cases switching between pectoral fin-based gaits
(Hale et al., 2006) before reaching a critical speed at which they
switch to body undulations with no pectoral fin movement.
Undulatory swimming speed is also frequency dependent while
amplitude may be a factor at slower speeds (e.g. Bainbridge, 1958).
While larval fish are known to have a discrete transition from
swimming with both pectoral fins and body undulations to
swimming with body undulations alone, the relationship of
kinematics to swimming speed up to that point is unknown but
The Journal of Experimental Biology 214, 3111-3123
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jeb.057497
RESEARCH ARTICLE
Movement and function of the pectoral fins of the larval zebrafish (Danio rerio)
during slow swimming
Matthew H. Green1, Robert K. Ho2 and Melina E. Hale1,2,*
1Committee on Computational Neuroscience, University of Chicago, Chicago, IL 60637, USA and 2Department of Organismal
Biology and Anatomy, University of Chicago, Chicago, IL 60637, USA
*Author for correspondence (mhale@uchicago.edu)
Accepted 5 June 2011
SUMMARY
Pectoral fins are known to play important roles in swimming for many adult fish; however, their functions in fish larvae are
unclear. We examined routine pectoral fin movement during rhythmic forward swimming and used genetic ablation to test
hypotheses of fin function in larval zebrafish. Fins were active throughout bouts of slow swimming. Initiation was characterized
by asymmetric fin abduction that transitioned to alternating rhythmic movement with first fin adduction. During subsequent
swimming, fin beat amplitude decreased while tail beat amplitude increased over swimming speeds ranging from 1.47 to 4.56
body lengths per second. There was no change in fin or tail beat frequency with speed (means ± s.d.: 28.2±3.5 and 29.6±1.9Hz,
respectively). To examine potential roles of the pectoral fins in swimming, we compared the kinematics of finless larvae generated
with a morpholino knockdown of the gene fgf24 to those of normal fish. Pectoral fins were not required for initiation nor did they
significantly impact forward rhythmic swimming. We investigated an alternative hypothesis that the fins function in respiration.
Dye visualization demonstrated that pectoral fin beats bring distant fluid toward the body and move it caudally behind the fins,
disrupting the boundary layer along the body’s surface, a major site of oxygen absorption in larvae. Larval zebrafish also
demonstrated more fin beating in low oxygen conditions. Our data reject the hypothesis that the pectoral fins of larval zebrafish
have a locomotor function during slow, forward locomotion, but are consistent with the hypothesis that the fins have a respiratory
function.
Supplementary material available online at http://jeb.biologists.org/cgi/content/full/214/18/3111/DC1
Key words: zebrafish, swimming, pectoral fin, genetic ablation, initiation.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

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important for understanding motor control in larvae and for
comparisons of motor systems among larvae, juveniles and adults.
Our second goal was to describe the kinematics in the initial phase
of swimming movement. Initiation of swimming that involves
pectoral fins and body undulations has not previously been
investigated in fish [but see the paper of Dubuc and colleagues
(Dubuc et al., 2008) for a review of lamprey swim initiation]. In
particular, we examined whether pectoral fin movement during
initiation demonstrates a distinct kinematic pattern and how the
movement coordination between the pectoral fins and between the
pectoral fins and the body is established. In humans, the initiation
of movement has been studied in depth and found to be remarkably
stereotyped (e.g. Carlsöö, 1966; Elble et al., 1994) and we were
particularly interested in whether such consistent patterns of
movement extended broadly to aquatic vertebrate swimming. In
addition to providing a better picture of fin usage in routine slow
swimming and the first examination of larval swim initiation, these
behavioral investigations provide necessary kinematic and
performance information for assessment of fin function.
Our last goal was to evaluate two general hypotheses that have
been put forth for the role of larval pectoral fin beating: that it
functions in locomotion (e.g. Batty, 1981) or that the behavior helps
to shed oxygen-depleted water surrounding the body (e.g. Hunter,
1972; Weihs, 1980; Osse and van der Boogart, 1999). Towards this
goal, we first compared the major components of routine swimming
between normal fish and age-matched finless zebrafish in which
the pectoral fins were genetically ablated through a morpholino
knockdown of the gene fgf24. We hypothesized that if the fins
function in locomotion, we would see decreased performance
and/or stability in finless fish or finless fish would have altered body
movements to compensate for the loss of the fins. Second, we
visualized flow patterns around the pectoral fins with dye to test
the hypothesis that the pectoral fins generate fluid movement that
might aid respiration by displacing the boundary layer along the
sides of the fish. Third, we examined swimming behavior of normal
fish in high and low oxygen environments to test the hypothesis
that decreased environmental oxygen results in increased pectoral
fin movement.
This research broadens our understanding of larval fish
locomotion by detailing the kinematics of the pectoral fins and the
body during the slow swimming bout and by addressing possible
functions of the pectoral fins in the movement of larvae. As the
zebrafish larva has become a model system for studying vertebrate
motor control and movement, these data provide a foundation for
work on the pectoral fin system of this model. Fish go through
remarkable post-hatching developmental changes in body
morphology, including size, and in physiology that significantly alter
how they interact with their physical environments. By addressing
hypotheses on the roles of larval pectoral fins, this work provides
an important step in understanding the functional development of
the pectoral fins.
MATERIALS AND METHODS
Animals
Embryos of wild-type zebrafish [Danio rerio (Hamilton 1822)] were
obtained from a laboratory breeding population. Embryos and larvae
were raised at 28.2°C in 10% Hank’s solution on a 14h:10h
light:dark cycle until filming at 5 days post-fertilization (d.p.f.).
To generate finless fish, a morpholino to the gene fgf24 was
injected into wild-type zebrafish embryos at the one- or two-cell
stage of development as previously described (Ahn et al., 2002;
Fischer et al., 2003). Phenol Red was co-injected to make it possible
to visualize the injection. At 48–72h post-fertilization, embryos that
had not yet hatched were dechorionated and fish without fins were
sorted from the rest of clutch. These fish continued to be raised
separately but under the same conditions as the un-injected fish.
There was no significant difference in total length (TL) between
normal and fgf24 morpholino-injected (finless) fish in our samples
(normal: 3.95±0.16cm; finless: 4.01±0.15cm; P>0.35) and the lack
of fins was the only difference between the groups that we could
discern through visual inspection of the animals.
For normal fish, our data set included 31 trials from 23 fish. So
as not to bias the data toward individuals represented by multiple
trials, we analyzed both the full data set and a more limited set that
included one trial per fish. The trial included was determined with
a random number generator. The data from finless fish include 22
trials from 13 fish and were analyzed in both full and culled sets.
Because the conclusions of statistical tests on the full data set did
not differ from those on the sets that included one trial per
individual, results from analysis of only the latter, reduced, data set
are presented unless otherwise noted.
High-speed video imaging of slow swimming
We used a Basler A500 (Basler Vision Technologies Inc., Exton,
PA, USA) high-speed digital video camera (maximum spatial
resolution of 5121280 pixels) mounted on a dissection microscope
(Leica Microsystems, Wetzlar, Germany) to film swimming bouts.
To study the detailed kinematics of slow swimming bouts, we filmed
groups of 5–6 normal or finless larval zebrafish in a rectangular
glass tank (x–y–z dimensions 6.531.5cm) at a frame rate of
1000Hz. To ensure that we filmed slow swimming bouts that were
not near the floor and sides of the tank, we recorded a focal plane
in the center of the tank. Individual fish were identified following
experiments using the unique skin pigmentation pattern on the head,
which could be seen in the video frames using ImageJ
(http://rsbweb.nih.gov/ij/). Trials in which fish identity was
ambiguous were not included in the analyses.
Visualization of flow near the pectoral fins
To visualize fluid motion near the pectoral fins, we placed a single
individual in a small Petri dish (3.5cm diameter, 1cm height) filled
with Hank’s solution and delivered a drop of Methylene Blue dye
(VWR International, West Chester, PA, USA; 0.5% diluted in
Hank’s solution) approximately 1mm anterior and 1–2mm lateral
to the head using a dulled syringe needle. The syringe was connected
via tubing to a pneumatic transducer (Fluke Corporation, Everett,
WA, USA), which allowed a very low pressure (~1mmHg) to drive
a slow leak of dye through the needle and into the water. For all
flow visualization experiments, fish were near the bottom of the
Petri dish and did not move forward during pectoral fin and body
movement, possibly because of surface friction or floor boundary
layer conditions. For a subset of flow visualization experiments,
fish were partially embedded in agar following methods described
previously (O’Malley et al., 1996) but with one pectoral fin free to
move in the surrounding fluid. This later test was performed to
address the possibility that fluid movement was induced by body
bending.
Imaging behavior at different dissolved oxygen
concentrations
To determine whether there was a change in pectoral fin or body
movement in low vs high dissolved oxygen (DO) concentration
Hank’s solution, we prepared low DO concentration Hank’s solution
by boiling, 500ml at a time, in a microwave for 20min. Boiled
M. H. Green, R. K. Ho and M. E. Hale
THE JOURNAL OF EXPERIMENTAL BIOLOGY