C H A P T E R
10
Respiratory System
Michael G. Jonz
Department of Biology, University of Ottawa, Ottawa, ON, Canada
Introduction
This chapter describes the general appearance and
morphology of the respiratory structures in zebrafish
and how they work together to form a functional
system. The structure and function of the respiratory
system in zebrafish is not particularly unique among
other cyprinid fishes. Therefore, where information
was not available in zebrafish, some of the information
contained within this chapter may be derived from
studies on other species.
For further information relating to the physiology of
gas exchange and respiratory physiology, the reader is
referred to the reviews cited in this chapter.
The Mechanics of Respiration
During ventilation, water first enters through the
mouth and into the buccal cavity, and then moves to
the opercular chambers across the gills and out through
an opening formed by each of the opercula (Fig. 10.1).
The operculum is a thin, bony flap of tissue that covers
and protects the entire gill complex on both sides. The
movement of water through the buccal and opercular
cavities and across the gills during ventilation is orches-
trated by a sequence of changes in volume and pressure
driven by a muscular pump (Moyes & Schulte, 2008).
The fish first creates a negative pressure within the
buccal cavity, drawing water in through the mouth
and oral valve. With the opercula closed, the opercular
cavities then begin to expand while positive pressure
is created in the buccal cavity by the closure of the oral
valve and buccal contraction. These forces lead to move-
ment of water in a caudal direction toward the gills.
Finally, the opening of the opercula and compression
of the opercular cavities project water through the gill
complex, and across the respiratory surface for gas
exchange, and out to the external environment. Debris,
The Zebrafish in Biomedical Research
https://doi.org/10.1016/B978-0-12-812431-4.00010-5
such as food or other particulate matter, may obstruct
the ventilatory cycle. However, the pharyngeal (or gill)
arches form gill rakers. These are small paired structures
that arise from the arches and are oriented toward the
buccal cavity, and help to filter out particles and prevent
them from becoming lodged within the gills.
The Gills
Morphology and Blood Flow
The gills are multifunctional organs that perform
many roles, such as osmoregulation, acid-base regula-
tion, excretion of nitrogenous waste, and gas exchange
(Evans, Piermarini, & Choe, 2005). For an exceptional
and unparalleled account of gill anatomy in fish, the
reader is referred to Hughes (1984). In teleost fish,
such as zebrafish, four gill arches are present on either
side of the animal and are oriented along the dorsoven-
tral axis. The gill arches are supportive structures and
are additionally important in respiration because they
deliver deoxygenated blood from the heart and ventral
aorta to the gill filaments via afferent branchial arteries;
they carry oxygenated blood from the gill filaments to
the dorsal aortae and systemic circulation via the
efferent branchial arteries.
The gill arches give rise to two rows of numerous gill
filaments that run perpendicular to the orientation of the
gill arches. Between each pair of filaments lies a small
interbranchial septum that extends from the gill arch
and attaches near the base of each filament. The septum
is the site of attachment of the adductor muscles that join
with the gill filaments. Contraction of the adductor
muscles draws together pairs of filaments and allows
for the rhythmic movement of the filaments (Hughes,
1984). The number and length of the gill filaments in
all teleosts is variable and continues to increase with
age. In mature zebrafish, the length of a single filament
103 © 2020 Elsevier Inc. All rights reserved.
104 10. Zebrafish Respiratory System
FIGURE 10.1 The mechanics of respiration in teleost fishes,
including zebrafish. Schema of the frontal section. Oral valves are
actually dorsal and ventral but are shown laterally for simplicity. Ar-
rows show the direction of water flow. Left: During inhalation, water
enters through the mouth and oral valve into the buccal cavity while
the opercula are closed. Right: The oral valve closes, the buccal cavity
contracts, and water passes into the opercular chambers, where the
gills are located, and through the open opercula. The opercular
chambers are located between the pharynx and opercula. After Wei-
chert, C. K. (1967). Elements of chordate anatomy. (3rd ed.). New York:
McGraw-Hill with permission from McGraw-Hill Education.
may typically measure 1e2 mm. The gill filaments may
sustain injury or infection in fish in the wild or captivity,
but recent evidence indicates that resected gill filaments
in zebrafish regenerate during the course of days or
weeks (Jonz, Zachar, Da Fonte, & Mierzwa, 2015). Deox-
ygenated blood from the gill arches enters the afferent
filament arteries of each filament, and the efferent
filament arteries return oxygenated blood to the gill
arches (Olson, 2002).
Each gill filament gives rise to numerous secondary
lamellae, which generally project in a dorsoventral
direction, perpendicular to the filaments. The lamellae
are evenly spaced, flattened structures that form small
channels between them, thus allowing for the flow of
water across their surface. The lamellae are thin-walled
and highly vascularized and are the site for gas
exchange in all water-breathing fish, including zebra-
fish. Blood flow throughout the lamellae runs in the
direction opposite to that of the flow of water over the
gills. This is called countercurrent flow, and efficiently
maximizes the exchange of O2 and CO2 across the
lamellar surface (Moyes & Schulte, 2008).
Many teleosts, including zebrafish, have a reduced
gill-like structure, called the pseudobranch, located ante-
rior to the first gill arch and embedded within the oper-
cular tissue (Jonz & Nurse, 2006; Laurent & Dunel-Erb,
1984). In zebrafish, the pseudobranch appears as a small
gill with fused filaments and lamellar-like structures. It
is only visible upon anterior dissection or removal of
the operculum. Since it lacks access to the external envi-
ronment and receives blood from the gills that has
II. Biology
already been oxygenated, the pseudobranch is thus not
believed to be involved in respiration. Although specu-
lative, the pseudobranch may act as a gland, sense
hypoxia, supply O2 to the retina, or participate in the
regulation of blood pH (Bridges, Berenbrink, Müller, &
Waser, 1998; Kern, Bösch, Unterhuber, & Pelster, 2002;
Laurent & Dunel-Erb, 1984).
Gill Development
In developing zebrafish, the pharyngeal arches first
produce gill filament primordia at 3 days postfertiliza-
tion (dpf; Kimmel, Ballard, Kimmel, Ullmann, &
Schilling, 1995; Jonz & Nurse, 2005). The filaments of
the gills in zebrafish develop primarily from the pharyn-
geal ectoderm (Hogan et al., 2004), although some struc-
tures internal to the gills may arise from the neural crest
(Mongera et al., 2013) and endoderm (Hockman et al.,
2017). At these early stages, the gills lack secondary
lamellae, and therefore, may not contribute significantly
to the gas exchange. The gills first become functional as a
respiratory organ at approximately 14 dpf (Rombough,
2002), and around this time begin to take on an adult-
like morphology, including the addition of secondary
lamellae (Shakarchi, Zachar, & Jonz, 2013). Before this
time, zebrafish larvae rely primarily on cutaneous respi-
ration for gas exchange (see below).
Internal Morphology
Gill filaments are supported by a cartilaginous rod in
order to provide structure or stiffness. Within the
lamellae lie the pillar cells, which extend lateral
processes toward one another to create a vascular cavity
for blood to flow (Wilson & Laurent, 2002). Pillar cells
may have a contractile function to control or redistribute
lamellar blood flow during respiration (Laurent, 1984).
The gill filaments and lamellae are covered by a thin
epithelium that lies atop a basal lamina and within which
reside numerous cell types that are critical for the func-
tionality of the gills (Laurent, 1984; Wilson & Laurent,
2002; Evans et al., 2005). The filament epithelium is non-
respiratory and is covered predominantly by cuboidal
and squamous pavement cells and mucous (goblet) cells.
Neuroepithelial cells (NECs) of the gill filaments are O2
chemoreceptors that detect changes in the environmental
or blood O2 and CO2/Hþ (Jonz, Fearon, & Nurse, 2004;
Qin, Lewis, & Perry, 2010), and thus lead to autonomic
responses, such as hyperventilation or changes in heart
rate, in order to maintain homeostasis.
The lamellar epithelium is particularly specialized for
respiration and gas exchange. It overlays the arterioarte-
rial circulation (Olson, 2002), and is generally two to
3 cell layers thick in most fish species (Wilson &
 Indicators of Stress to the Respiratory System
Laurent, 2002). The cell types found in the lamellar
epithelium include the cuboidal and squamous pave-
ment cells, undifferentiated cells that contact the basal
lamina, and some mucous cells (Evans et al., 2005;
Wilson & Laurent, 2002). Neuroepithelial cells have
also been found in the lamellae of zebrafish (Jonz &
Nurse, 2003), although a role in O2 sensing for these cells
has still not been established.
Neurobiology
The gill is a highly perfused organ and is a major site
of circulatory and vasomotor control (Nilsson & Sundin,
1998). Such control is mediated by the nervous system
and is critically important in regulating respiratory func-
tion. Innervation of the gills by the cranial and spinal
nerves has been described in teleost fish, including
zebrafish (Nilsson, 1984; Sundin & Nilsson, 2002; Jonz
& Nurse, 2008; Jonz & Zaccone, 2009). As in other
species, the gills are innervated by the nerve fibers of
the cranial nerves IX (the glossopharyngeal nerve) and
X (the vagus nerve), and by fibers originating from the
sympathetic chain and entering the cranial nerve trunk
via gray rami communicantes (Fig. 10.2). Each gill arch
is innervated by two nerve rami through which all nerve
fibers are delivered: a pretrematic ramus that lies
FIGURE 10.2 Simplified scheme of innervation to the gill region in
teleost fish. The gills are innervated by nerve fibers from the cranial
nerves, and by fibers from the sympathetic chain that enter the cranial
nerve trunk via gray rami communicantes. Each gill arch is innervated
by a pretrematic ramus (anterior to the gill slit) and a posttrematic
ramus (posterior to the gill slit) Pretrematic rami carry sensory fibers,
while posttrematic rami carry sensory and motor fibers. Reprinted from
Jonz M. G., Zaccone, G. (2009). Nervous control of the gills. Acta Histo-
chemica, 111 207e216 with permission from Elsevier.
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105
anterior to the gill slit (the space between gill arches),
and a posttrematic ramus that lies posterior to the gill
slit. Pretrematic rami carry the sensory (afferent) fibers,
while posttrematic rami carry the sensory and motor
(efferent) fibers. Chemoreceptive NECs of the gill
filaments receive sensory innervation via the cranial
nerves IX and X and convey information, such as
low-O2 status (hypoxia) of the environment, to the
central nervous system (Jonz & Nurse, 2003).
Cutaneous Respiration
In many anamniotic vertebrates, such as fish, the skin
is an important site of gas exchange during early devel-
opmental stages (Rombough, 1988). In developing
zebrafish, cutaneous respiration accounts for nearly all
gas exchange and does not become limiting until
approximately 10 dpf (Rombough, 2007), when the gills
are fully functional. Moreover, there is evidence that the
pectoral fins in developing zebrafish contribute to respi-
ration by actively moving O2-depleted water away from
the body surface and pulling in O2-rich water toward the
body surface, thereby enhancing gas exchange (Hale,
2014). Cells with chemoreceptor activity have also
been described in the skin of zebrafish that detect
changes in O2 and elicit hyperventilatory responses to
hypoxia (Coccimiglio & Jonz, 2012).
Indicators of Stress to the Respiratory System
Hyperventilation
All fish hyperventilate in response to reduced O2
availability (Perry, Jonz, & Gilmour, 2009). Hyperventi-
lation in fish is described as an increase in breathing fre-
quency, as can be visually observed by an increased rate
of movement of the mouth and opercula. Fish may also
produce a hyperventilatory response to high levels of
water CO2 or Hþ (Gilmour, 2001). In zebrafish, these re-
sponses have been shown to be mediated by O2-chemo-
receptive NECs of the gill filaments (Abdallah, Jonz, &
Perry, 2015a; Jonz et al., 2004; Porteus et al., 2014; Qin
et al., 2010). A typical resting ventilatory rate for adult
zebrafish is approximately 160 breaths min1; whereas
hypoxia (PO2 of 35e40 mmHg) may elevate ventilation
frequency to above 300 breaths min1 (Jonz & Nurse,
2005; Vulesevic, McNeill, & Perry, 2006). Adult zebrafish
will begin to display an increase in ventilation frequency
under mild hypoxia at PO2 of approximately 110 mmHg
(Vulesevic et al., 2006), where normal PO2 would be
approximately 150 mmHg.
In developing zebrafish, the hyperventilatory
response to hypoxia is not mature but begins to appear
106 10. Zebrafish Respiratory System
at approximately 7 dpf, when the gill chemoreceptors
become innervated (Jonz & Nurse, 2005). Before this
time, zebrafish display an early behavioral response to
hypoxia that involves an increase in whole-body move-
ment (Coccimiglio & Jonz, 2012). Compared to adults,
zebrafish larvae display lower ventilation frequencies.
Although variable, resting ventilation frequency is typi-
cally 50e100 breaths min1 in zebrafish larvae, with
responses to hypoxia measuring at about 150 breaths
min1 (Rahbar, Pan, & Jonz, 2016).
Aquatic Surface Respiration
Under severe hypoxic stress, in addition to hyperven-
tilation, fish display aquatic surface respiration (ASR)d
a behavior in which the fish seeks well-oxygenated wa-
ter by moving up to the air-water interface (Chapman &
McKenzie, 2009; Perry et al., 2009). Zebrafish may begin
to engage in ASR as PO2 reaches 50 mmHg or lower,
although a threshold PO2 of 30 mmHg was calculated
as the level below which zebrafish will rely on ASR for
survival (Abdallah, Thomas, & Jonz, 2015b). Decreasing
PO2 results in an increased amount of time that zebrafish
will spend performing ASR. Evidence also indicates that
as zebrafish are acclimated to hypoxia, the ASR
threshold will decrease, meaning that zebrafish accli-
mated to hypoxia are able to postpone performing
ASR until more severe levels of hypoxia. ASR is evident
in zebrafish as early as 5 dpf, and larvae display an ASR
threshold of 16 mmHg.
Conclusion
This chapter has outlined the important structures of
the respiratory system of zebrafish for the researcher or
student interested in using zebrafish as a model organ-
ism in biological or biomedical research. The mecha-
nisms of the buccal and opercular cavities, together
with the structure of the gills, were described to provide
an anatomical overview of the important structures of
the respiratory system. In zebrafish, in particular, infor-
mation regarding the role of chemoreceptors and the
nervous system in mediating responses to hypoxia,
and identification of behavioral indicators of stress,
were described in order to help researchers and animal
care personnel recognize when the zebrafish respiratory
system is under stress. The zebrafish respiratory system
is very much like that of other related fish species, and so
the interested reader will have access to a vast literature
base for further reading.
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