C H A P T E R

32
Zebrafish Larviculture
Christian Lawrence
Boston Children’s Hospital, Boston, MA, United States of America

Introduction in shallow, still, or slow-moving water in and around

the margins of water bodies (Roy, Shukla, & Bhat,
The practice of larviculture is a critical part of any 2017). This level of habitat diversity, coupled with the
research program that utilizes the zebrafish model monsoonal climate regime that defines this region,
system, and it remains one of the great operational chal- necessitates that, like many animals that inhabit this
lenges in fish husbandry. It is also one of the first barriers part of the world, the zebrafish are able to tolerate
to entry into zebrafish research, as an inability to grow wide and sometimes rapid alterations in environmental
animals will either severely limit the type and scale of conditions. Indeed, zebrafish can be considered exem-
science possible, or preclude it altogether. plary in this regard, having been documented in
Relative success in larval rearing is defined by growth habitats that experience wide temporal fluctuations
and survival rates, with the goal being the maximization in salinity, temperature, pH, and other chemical and
of both values, not only for the sake of operational effi- physical parameters (Arunachalam et al., 2013;
ciency but also for animal welfare. Compared to many Engeszer et al., 2007; McClure, McIntyre, & McCune,
fish species commonly cultured in captivity, zebrafish 2006; Spence et al., 2006).
are not particularly difficult to rear, but their small size
and limited swimming ability at the onset of first feeding
are among the challenges that must be addressed. Natural History and Reproductive Biology
Protocols for zebrafish larviculture must incorporate an In the wild, zebrafish display a range of social behav-
understanding of (1) the natural history, biology, behavior, iors and tend to associate in groups that vary in size and
and nutritional requirements of the developing fish, and activity in accordance with the habitat type (Roy et al.,
(2) the relationship between these traits and the physical 2017; Suriyampola et al., 2015, p. 1159). They occupy
and chemical properties of water. The best and most effec- the entirety of the water column but more commonly
tive larval rearing techniques strike the proper balance inhabit shallower waters along banks and margins, often
between the two and require vigilant, attentive caretakers in association with overhanging vegetation (Spence et al.,
with keen observational skills to administer them. 2006). They are omnivorous, opportunistic feeders,
consuming small zooplankton, terrestrial, and aquatic in-
sects, and even plant material (Arunachalam et al., 2013;
Natural History/Biology of Larvae
McClure et al., 2006; Spence, Fatema, Ellis, Ahmed, &
Smith, 2007a). Gut content analyses reveal that terrestrial
Natural Distribution insects, presumably falling into the water from over-
Zebrafish are native to south Asia and are widely hanging vegetation, are the predominant prey type
distributed across much of India, Bangladesh, and low- (Arunachalam et al., 2013), but this may simply reflect
land Nepal (Arunachalam, Raja, Vijayakumar, relative abundance in the environment at a given time
Malaiammal, & Mayden, 2013; Engeszer, Patterson, point.
Rao, & Parichy, 2007; Spence, Gerlach, Lawrence, & Spawning commences prior to the onset of the rainy
Smith, 2008; Talwar & Jingran, 1991). While these fish season, with the fish breeding in pairs or small groups
may be found in virtually any freshwater habitat type in shallow water in and around the margins of water
within this vast region (streams, rivers, rice paddies, bodies they inhabit (Hutter, Penn, Magee, & Zala,
ponds, lakes, etc.), they most commonly tend to occur 2010; Spence, Gerlach, Lawrence, & Smith, 2008).

The Zebrafish in Biomedical Research

https://doi.org/10.1016/B978-0-12-812431-4.00032-4 365 © 2020 Elsevier Inc. All rights reserved.
366 32. Zebrafish Larviculture

Fertilization is external; and during spawning, the Development rapidly proceeds over the next several
nonadhesive, demersal eggs are scattered and fall to weeks of life. Fish are classified as larvae up until the
the substrate with no parental care. In the wild, female larval fin fold disappears, and they attain a complete
zebrafish have been shown to prefer to oviposit in scaling pattern (Parichy et al., 2009). This corresponds
shallow water among submerged vegetation and gravel to an SL of w11.0 mm and may take several weeks to
(Spence, Ashton, & Smith, 2007c), presumably because several months to attain (Augustine, Gagnaire, Floriani,
these materials provide protective cover for developing Adam-Guillermin, & Kooijman, 2011).
embryos (Engeszer et al., 2007). The embryos hatch
within a few days, and spend the first few weeks of
life in shallow, nutrient-rich habitats, feeding on small Feeding
zooplankton and other organisms within the water
column. Meeting the nutritional demands of larval fish is an
age-old challenge in aquaculture (Cahu & Infante,
2001; Hamre et al., 2013; Onal & Langdon, 2005), and
Biological Characteristics and Staging of the selection and administration of the diet to animals
Zebrafish Larvae during this time period will define the success or failure
of any rearing program. There are a number of different
There has been considerable discussion in the litera- factors that must be considered, the most important of
ture regarding the appropriateness of defining age clas- which will be treated below, with a specific relationship
ses in developing zebrafish based on the time since their to zebrafish.
fertilization versus distinct morphological criteria/size
(Kimmel, Ballard, Kimmel, Ullmann, & Schilling, 1995;
Parichy, Elizondo, Mills, Gordon, & Engeszer, 2009).
From the moment of fertilization, a whole host of envi- Nutrient Requirements
ronmental conditions influence the rate of development, After hatching, larval zebrafish subsist entirely off the
so the further one goes out from time zero, the less rele- nutritional reserves provided within the yolk (Hanisch,
vant that time point becomes in the defining stages. Küster, Altenburger, & Gündel, 2010). Larvae will not
The exception to this is during the first 24e72 h of life, feed until uptake and process of external nutrients
if temperature, medium, density, and photoperiod are become possible, a stage which is coincident with suffi-
standardized (Kimmel et al., 1995). Once the fish inflates cient development of a functional digestive system (Ng
its swim bladder and begin to swim and feed, it is more de Jong Curtain, Mawdsley, White, Shin, Appel, Dong,
appropriate to use standard length (SL). The size ranges et al., 2005) and the ability to regulate their position
given below are based on those given by Parichy et al. in within the water column (Strähle et al., 2012). The
their classic paper defining postembryological staging confluence of these developmental events is the swim-
in zebrafish (Parichy et al., 2009). up period when larvae inflate their gas bladders and
The larval stage begins at 48e72 h postfertilization begin feeding on exogenous prey (Strähle et al., 2012).
(hpf), when the embryos hatch from the chorion, which Zebrafish larvae should be presented with food
is progressively weakened by release of a hatching immediately after this point because any energy remain-
enzyme and spontaneous flexing movements of the ing in their yolks is rapidly depleted once the animals
embryo (Laale, 1977). Over the next several days, start actively swimming. Still, it is important to consider
the developing larvae show low levels of spontaneous that there is some overlap (w24e48 h depending on
activity but slowly begin to make their way closer to temperature and activity rates (Jardine & Litvak, 2003;
the surface, usually by “creeping” their way up Semova et al., 2012)) between total yolk absorption and
submerged structures by means of specialized adhesive the commencement of independent feeding. This pro-
cells on the epidermis of the head (Laale, 1977). vides the fish with a nutritional head-start during a
Once they reach an SL of 3.4e3.7 mm (mm), which critical period when their locomotor capacity is at its
usually occurs within 48 h after hatching, the larvae lowest and/or if suitable prey is scarce (Lawrence,
will swim up to the air-water interface and inflate their et al., 2012a). If fish are not fed at all, they will starve
gas bladders by swallowing air and forcing it into their to death within a few days after swim-up, the timing
guts (Goolish & Okutake, 1999). This developmental of which depends upon temperature and other environ-
event, which allows them to regulate their position mental factors. For example, zebrafish that are never
within the water column, is coincident with the onset provided with feed will starve by 10 days postfertiliza-
of both exogenous feeding and active avoidance behav- tion (dpf) when maintained at 28
Centigrade (Jardine
iors (Kimmel et al., 1995; McHenry Feitl, Strother, & Van & Litvak, 2003).
Trump, 2009; Strähle et al., 2012).

III. Husbandry
 Feeding 367
During the first few weeks of life, larval zebrafish expanding their mouth cavity. This action creates a
need to feed continuously to fuel rapid growth (Hamre strong flow of water that draws the item into the mouth
et al., 2013), especially the structural remodeling to be consumed (Bianco, Kampff, & Engert, 2011; China
required for the transformation from the larval to the & Holzman, 2014; McElligott & O’Malley, 2005).
juvenile stage (Parichy et al., 2009; Rombough, 2002). Given how this mechanism works, the size of item
Prey items need to be highly digestible, as the larval relative to the size of the fish is important, and the size
alimentary tract is functional, but structurally simple of the particle that fish are able to consume is defined
with a limited capacity for storage and assimilation of by the width of their mouths. This metric increases
complex molecules (Field, Roeser, Stainier, Ober, 2003; rapidly as the fish grows. For example, in one study,
Govoni, Boehlert, & Watanabe, 1986; Ng et al., 2005; 3.8 mm fish had a mean mouth width of 180e200 mm
Zambonino-Infante et al., 2008). The ability of the fish (micrometers), while larvae that were 5.2 mm had a
to break down and assimilate more complex nutrients mean mouth width of 290e320 mm (Önal & Langdon,
increases as they mature, but in general, dietary items 2016). It is important to understand that these values
should be as digestible as possible to maximize do not necessarily correspond to the actual size limit
efficiency, and therefore, growth rates. of what a fish of a particular age and/or length can
As is the case with many other fish species, the pre- consume. This is more accurately delineated by the ratio
cise nutrient requirements of zebrafish larvae have not of particle size to mouth width (Önal & Langdon, 2016).
been formally delineated (Watts, Powell, & D’Abramo, The upper limit of this value in developing zebrafish
2012). However, limited inferences about this can be larvae has been shown to be in the range of 40%e55%,
made upon the basis of growth and survival perfor- but the fish actually tend to consume much smaller
mance of the fish when reared on different diets. Indeed, items, more in the range of <20% (Önal & Langdon,
zebrafish appear to perform best when first presented 2016). Probably, this is because they have higher success
with live zooplankton that mimics their diet in natural rates in feeding on smaller items than items that are at
settings (Lawrence, 2007; Spence et al., 2008). the upper limit of their abilities. As they continue to
While there are a number of factors that contribute to grow, fish will selectively feed on increasingly larger
this success, it is probably due at least in part to the basic particles, and once they transition to the juvenile stage,
nutritional characteristics of these feeds. Commonly they become more effective at feeding first at the surface
cultured zooplankton species like brine shrimp (Artemia and then finally in the benthos (Harper & Lawrence,
sp.) or rotifers (Brachionus sp.) generally possess basic 2010).
nutritional characteristics that support larval fish devel-
opment, like high protein content and higher digestibil-
ity; but it is important to consider that they will vary in
nutritional composition depending on culturing condi-
Feed Characteristics
tions and feed inputs (Lavens & Sorgeloos, 1996a). Prey items must be both available and attractive to
Rotifers, Artemia, and other zooplankton species may larval fish. Availability is a key parameter; larvae must
be “bioencapsulated” to boost their profiles of essential be able to locate and subsequently consume items while
fatty acids, key amino acids, vitamins, and minerals expending minimal energy to do so. For many species
known to be important for fish growth and survival that are obligate water column feeders early in develop-
(Hamre, Mollan, Sæle, & Erstad, 2008; Figueiredo, van ment, including zebrafish, this is one reason why live
Woesik, Lin, & Narciso, 2009; Ritar et al., 2004; Thépot zooplankton is so effective; it is biologically available
et al., 2016). This approach has been successfully applied to larvae because when alive, the organisms swim
to zebrafish larviculture (Best, Adatto, Cockington, actively throughout the water column and therefore
James, & Lawrence, 2010; Lawrence et al., 2016). are available for the fish to encounter (Conceição,
Yúfera, Makridis, Morais, & Dinis, 2010). For the same
reason, processed feeds, like microdiets, are designed
to maximize their residence time in the water so that
Feeding Behavior/Anatomy
larva may encounter them. Considerable attention is
As adults, zebrafish feed effectively at the surface, devoted to sinking rates, retention of water-soluble
benthos, and within the water column (Lawrence, nutrients, as well as physical characteristics and mecha-
2007). As first-feeding larvae, however, they are essen- nisms to keep them in suspension (Bonaldo, Parma,
tially restricted to items within the water column, due Badiani, Serratore, & Gatta, 2011; Onal & Langdon,
mostly to their limited swimming ability at this stage 2005; Person, 1989). Sinking rates also can apply to
of development (Harper & Lawrence, 2010). Larval zooplankton in some situations; for example, some spe-
zebrafish are suction feeders and capture prey by swim- cies only persist for finite periods once introduced to
ming up to it and opening their mouths while culturing tanks (i.e., Artemia in freshwater).

III. Husbandry
368 32. Zebrafish Larviculture

Another aspect of feed availability is the rate at which to fish larvae than are inanimate processed particles.
the particle moves in the water. This applies especially to Among the species of zooplankton most commonly fed
live feeds, as zooplankton swim in different ways and at to laboratory zebrafish, Artemia sp. probably elicit the
different speeds (Lavens & Sorgeloos, 1996a). This must greatest response based on their swimming behavior,
be matched with the swimming ability and age of the which is more erratic and faster than rotifers or Parame-
target fish. For example, zebrafish larvae are too slow to cium, both of which move more slowly through the
efficiently capture Artemia nauplii during the first few water column (Best et al., 2010; Harper & Lawrence,
days of exogenous feeding and are much more adept at 2010). Processed feeds having superior buoyancy when
feeding on slower moving Paramecium (Westerfield, combined with favorable upwelling flow regimes would
2007) or rotifers during that period (Best et al., 2010; Law- mimic the random movement that allows fish to locate
rence, James, & Mobley, 2015; Lawrence et al., 2016). As Artemia while feeding.
they grow, they more effectively feed on the larger, faster
Artemia nauplii (Harper & Lawrence, 2010). Processed
feeds will also move throughout the water column at
different rates depending on their size, density, and
Feed Types
composition (Girin, Halver, & Tiews, 1979; Kolkovski, Both live and processed feeds can be utilized as prey
2013). As the fish grow and develop their ability to ma- items for first feeding zebrafish. Among live feeds, three
neuver directionally, this becomes of less concern. species predominate in their application across the
The attractiveness of a given feed item is also critical, zebrafish research community. Paramecium sp. were
as larval fish are not indiscriminate. In fact, larvae selec- among the first live feeds that pioneering zebrafish lab-
tively track and consume prey based on a wide variety oratories used to rear larval fish (Nüsslein-Volhard
of characteristics. For example, fish display color prefer- Dahm, 2002; Westerfield, 2007) and are still commonly
ences from a young age, and this will often extend to used in the field (Varga, 2011). Paramecium are ciliated,
prey selectivity (Browman & Marcotte, 1987; El-Sayed, unicellular, multi-nucleated organisms that reproduce
El-Ghobashy, & El-Mezayen, 2013). Zebrafish show an both asexually and sexually, depending upon environ-
innate preference for red food as adults (Spence & mental conditions. Under ideal conditions, they can
Smith, 2008). This has not been tested in fish during reproduce asexually 2e3 times daily, and they feed pri-
the larval stage, but it is thought that the preference marily on bacteria, as well as microalgae, yeast, and
observed in adults reflects that the coloring of small protozoa (ALLEN & NERAD, 1978; Beale, Preer,
zooplankton in wild habitats is often red or orange & Harumoto, 2008; Sonneborn, 1970). Depending on
(Spence et al., 2007a; Spence & Smith, 2008). the species, Paramecium may range in size but on
The smell and taste of a feed are also components of its average measure about 50  180 mm (width by length),
attractiveness. The olfactory system in zebrafish is func- which is suitable for first feeding zebrafish (Varga,
tional from swim-up (Hansen & Zeiske, 1993; Miyasaka 2011). They can be cultured on various diets, but proto-
et al., 2013), indicating the important role that it plays cols utilizing yeast or LB media are most common
in helping fish to find and detect prey. Larvae can detect (Nüsslein-Volhard Dahm, 2002; Varga, 2011; Westerfield,
amino acid mixtures in the water as early as four dpf 2007). Paramecium swim slowly throughout the water
(Lindsay, 2004), and they can utilize this sensitivity, in column and may be easily captured by larvae that
combination with the directional sensing of currents by encounter them. In addition to that fact that they may
the lateral line (Rønnestad et al., 2013), to help locate be readily acquired and cultured, these traits have made
food items in the water column. Once prey is located, them a favorite diet for ornamental fish growers (Mitchell,
fish use their taste buds to determine its palatability 1991) and often the primary component of “infusoria”
(HARA, 1994; Rønnestad et al., 2013). Functional taste used for rearing fish in home aquaria (Mukai, Sani, &
bud cells are also present in zebrafish at the onset of inde- Kadowaki, 2016). Numerous reports demonstrate that
pendent feeding (Hansen, Reutter, & Zeiske, 2002), and they are effective prey items for first feeding larval zebra-
so it is reasonable to presume that taste plays a role in fish, especially for the first several days postswim-up
prey selectivity in this species, even at a young age. (Biga & Goetz, 2006; Goolish, Okutake, & Lesure, 1999;
Another part of the attractiveness of a feed particle is Hensley & Leung, 2010; Mullins, Hammerschmidt,
the manner, as opposed to the rate, in which it moves Haffter, & Nüsslein-Volhard, 1994; Nüsslein-Volhard
through the water. Prey items that have active, irregular Dahm, 2002; Wilson, 2012), although there are some indi-
swimming motions are thought to be more conspicuous cations that they may not adequately meet nutritional de-
to fish larvae (Buskey, Coulter, & Strom, 1993). For mands of the fish much beyond this stage (Lawrence,
example, copepods (e.g., Arcatia sp.) are particularly 2007; Wilson, 2012).
well known in this regard (Conceição et al., 2010). Rotifers, most especially the euryhaline Brachionus
In this way, zooplankton are generally more attractive plicatilis or B. rotundiformis, are the most important live

III. Husbandry
 Feeding 369
prey for many cultured fish species during the first zebrafish culture, for the entire life cycle (Carvalho,
feeding phase, due to their small size, slow swimming Araujo, & Santos, 2006; Gonzales & Law, 2013; Lawrence,
behavior, and their amenability to intensive culture Best, James, & Maloney, 2012c, 2016; Markovich, Rizzuto,
and bioencapsulation with nutrients essential for & Brown, 2007). They may be used as a prey item for first
growth and development (Cahu & Infante, 2001; Lavens feeding zebrafish (Carvalho et al., 2006; Conceic, 2010),
& Sorgeloos, 1996b; Lawrence, Sanders, & Henry, although this method can be challenging to reproduce
2012b). The freshwater rotifer B. calyciflorus is also because the larvae do not efficiently capture the larger
utilized because of its similar attributes but is less com- and fast swimming nauplii (Lawrence et al., 2015). This
mon due to the fact that it can be more challenging to difficulty is further compounded by the fact that Artemia
culture than its euryhaline relatives (Aoyama et al., may only survive in freshwater habitats for a short period
2015; Arimoro, 2006). Like Paramecium, brachionid roti- of time, reducing their bioavailability to larval fish.
fers asexually reproduce when conditions are favorable, A number of other zooplankton species may be used
and switch to sexual reproduction when resources as a prey item for larval zebrafish. These include ciliates
become limited (Lavens & Sorgeloos, 1996a; Lawrence (Tetrahymena sp.) (Kopp, Legler, & Legradi, 2016;
et al., 2016). The goals of typical batch or continuous Rendueles et al., 2012) and microworms (e.g., Panagrellus
culturing methods for rotifers center around promoting redivivus) (Amaral & Johnston, 2012; Westerfield, 2007).
favorable environmental conditions to promote Other zooplankton, like cladocerans (water fleas), cope-
maximal population growth through asexual reproduc- pods, and krill, are too large for larval zebrafish, and are
tion (Lawrence et al., 2012b, 2016). “Type L” B. plicatilis only suitable for fish from the subadult stage or beyond.
range in size from w130 to 340 mm, while “type S” Larval fish may also be reared on processed diets,
B. rotundiformis are w100e210 mm; both feed primarily either exclusively, or in tandem with live feeds. The chal-
on small phytoplankton (Lavens & Sorgeloos, 1996b). lenges associated with feeding small, first feeding fish
The freshwater B. calyciflorus are similar in size, larvae with processed diets are well-documented (Cahu
w260 mm (Aoyama et al., 2015). The success of brachio- & Infante, 2001; Girin et al., 1979; Hamre et al., 2013;
nid rotifers as a first prey item for larval zebrafish has Kolkovski, 2013; Onal & Langdon, 2005; Person, 1989),
been well documented, with numerous publications primarily because many species of fish are designed
reporting high rates of growth and survival in zebrafish by nature to feed on zooplankton, and all of the inherent
fed on both euryhaline and freshwater species (Aoyama features of these organisms are difficult to incorporate
et al., 2015; Best et al., 2010; Farias & Certal, 2016; into an inanimate particle. Among the most notable
Lawrence et al., 2016; Siccardi et al., 2009). problems associated with processed “micro” diets is
The brine shrimp, Artemia salina (and other species, leaching of water-soluble nutrients upon hydration
including A. franciscana) is the most important and prev- (Yúfera, Kolkovski, Fernández-Dıaz, & Dabrowski,
alent live prey for commercially produced species of fish 2002; Kvåle et al., 2007), a situation made more difficult
and shellfish (Lavens & Sorgeloos, 1996b). This small by the very high surface area to volume ratios of small
branchiopod crustacean, which is found in hypersaline particles, especially those under 100 mm. Diets may be
lakes and lagoons in several locations globally, produces microencapsulated in such a way to minimize these los-
dormant embryos or “cysts” that can withstand desicca- ses (Yúfera, Pascual, & Fernández-Dı́az, 1999), but this
tion in dry lake beds. These cysts can be harvested, pro- remains a problem, especially since residence time in
cessed, and stored such that they can be later the water must be maximized in order for weakly
“reanimated” on demand by hydration in seawater, to swimming fish larvae to be able to encounter them.
hatch into free-swimming nauplii that can be used as a The other issues associated with these diets include
food source for larval fish and shellfish. Artemia are easily precise nutrient formulation, palatability, color, and
cultured in batches, and a typical production cycle movement in the water. Live feeds are superior in
(hatching from cysts into stage 1 nauplii) takes w24 h most if not all of these areas.
(Treece, 2000). The first instar nauplii at w400e500 mm Despite these shortcomings, it is possible to rear fish
in length (Harper & Lawrence, 2010; Lavens & Sorgeloos, exclusively on these diets, and this extends to zebrafish
1996b) are a suitable live prey source for a variety of first (Harper & Lawrence, 2010). First feeding zebrafish will
feeding fish. After hatching, the nauplii will eventually readily consume inanimate particles (Onal & Langdon,
molt into the second stage larval metanauplii, which is 2000; Önal & Langdon, 2016), and there are numerous
at that point capable of exogenous feeding. From then examples in the literature of them being reared on pro-
on, the Artemia must be fed, or “bioencapsulated,” with cessed diets alone (Carvalho et al., 2006; Farias & Certal,
microalgae or fatty acid emulsifications to maintain their 2016; Goolish et al., 1999; Hensley & Leung, 2010).
nutritional value for the fish (Figueiredo et al., 2009; Sor- It should be noted that survival and or growth is typi-
geloos, Dhert, & Candreva, 2001). Artemia are perhaps the cally reduced when compared to performance on live
most widely utilized feed itemdlive or processeddin

III. Husbandry
370 32. Zebrafish Larviculture

diets, however (Carvalho et al., 2006; Farias & Certal, farmed fish species (Almazán-Rueda, Van Helmond,
2016; Goolish et al., 1999). Verreth, & Schrama, 2005; Boeuf & Falcón, 2001; Boeuf
& Le Bail, 1999; El-Sayed & Kawanna, 2004; Mclean,
Cotter, Thain, & King, 2008; Ullmann et al., 2011).
Water Quality There are a growing number of studies on how
various aspects of light impact zebrafish embryos and
The physical and chemical characteristics of water larva. For example, the ambient color spectrum was
exert a broad range of effects on larval survival. For found to exert profound effects on embryo hatching
any species, there is a given set of parameters that rates, larval growth and survival in zebrafish, with fish
must be maintained within a certain range that will pro- exposed to blue and white light showing highest rates
mote optimal performance. of growth and survival along with the lowest rates of
malformation (Villamizar, Vera, Foulkes, & Sánchez-
Vázquez, 2013). In the same study, the authors found
that exposure to constant light depressed growth and
Physical reduced survival, while fish that were reared in constant
A number of physical factors impact the behavior, darkness showed 100% mortality by 18 days posthatch.
biology, growth, and survival of larval fish as they However, it has also been shown that larval zebrafish are
develop. Of all of these factors, the temperature is able to forage in the dark using the lateral line system
perhaps the most significant and pervasive as it affects (Carrillo & McHenry, 2016), suggesting that the negative
both the physiology of the fish and the physics of the results in some studies might be related to feed type or
water (von Herbing, 2002). As poikilotherms, fish are availability.
profoundly sensitive to temperature. While zebrafish Interestingly, manipulation of photoperiod (i.e., sub-
are among the most eurythermal fish species on record, jecting fish to extended or continuous light conditions)
with an acclimated thermal tolerance of 6.7e41.7
C has been utilized in a number of aquaculture species
under controlled conditions (Cortemeglia & Beitinger, with some success (Boeuf & Falcón, 2001; Partridge
2005; Schaefer & Ryan, 2006), temperature exerts myriad et al., 2011; Valenzuela et al., 2012), presumably due to
changes on gene expression and physiology, especially increased feed intake and feed conversion. Certainly,
during early life (von Herbing, 2002; Long et al., 2013; this could be the case for zebrafish, but none of the
Villamizar, Ribas, Piferrer, Vera, & Sánchez-Vázquez, studies published so far in the literature of fish reared
2012; Scott & Johnston, 2012). under extended daylight have evaluated performance
In general, the range within which embryonic devel- in the presence of unlimited prey. At least in one study,
opment proceeds normally in zebrafish is between 24 a higher proportion of malformations was observed
and 32
C (Schirone & Gross, 1968). The thermal optima when larvae were exposed to constant light (Villamizar
for zebrafish in controlled conditions have not been et al., 2013). It is critical to consider that any artificial
formally defined, but recommendations can be found manipulation of photoperiod and lighting should be
in the literature (e.g., the 24e30
C range promoted by done in the context of the visual physiology, biology,
Matthews et al. (Matthews, Trevarrow, & Matthews, and ecology of the target species.
2002)) and generally conform to the biological data. The color of housing enclosures has also been shown
The widely cited maintenance temperature of 28.5
C to influence growth performance in a number of aquacul-
(Westerfield, 2007) is derived from the classic embryo- ture species (Imanpoor & Abdollahi, 2011; Mclean et al.,
logical staging experiments of Kimmel et al (Kimmel 2008; Ullmann et al., 2011; Yasir & Qin, 2009). A primary
et al., 1995), and this does not necessarily represent an causative factor for this phenomenon is thought to be
optimum with respect to performance. However, some contrast created between differential background colors
existing data supports the idea that zebrafish growth is and prey types; background colors that enhance this
maximized around this value; in one study, fish grown contrast are thought to improve capture/ingestion rates.
at 28
C were larger than cohorts reared under 22, 25, However, it is critical to note that this relationship is
or 31
C regimes (Sfakianakis, Leris, Laggis, & Kentouri, entirely dependent upon species. For example, spotted
2011). Villamizar et al. observed a similar trend in their grouper reared in white tanks ingested more rotifers
study on daily thermocycles in zebrafish (Villamizar and showed better growth than those grown in dark en-
et al., 2012). closures (Zhang et al., 2015). Striped trumpeter larvae
Light is another major physical factor that impacts showed enhanced growth and food consumption when
larval fish on many levels. As a physical factor, light grown with live feeds in dark backgrounds (Cobcroft,
encompasses color spectrum, photoperiod, and intensity; Shu-Chien, Kuah, Jaya-Ram, & Battaglene, 2012).
and its effects on larval growth, development and other The variability shown in this response across species
performance metrics have been extensively evaluated in and situation illustrates the complex relationship that

III. Husbandry
 Water Quality 371
exists between color spectrum, light intensity, and prey products, particularly unionized ammonia (NH3) and
type in larval fish. nitrite (NO-2). There are numerous examples of the
The effect of tank color has not been well explored toxicity of these compounds to developing fish
relative to zebrafish growth performance. However, (Adelman, Kusilek, Koehle, & Hess, 2009; Barbieri &
tank background colors have been shown to impact Doi, 2012; Bardon-Albaret & Saillant, 2016; Daoust &
zebrafish behavior (Blaser & Rosemberg, 2012; Vignet Ferguson, 1984; Jensen, 2003; Palachek & Tomasso,
et al., 2013). In one study, tank wall color significantly 1984; Rodrigues, Schwarz, Delbos, & Sampaio, 2007;
affected depth preference in adult fish (Blaser & Gold- Wang, Wang, Yu, & Jiang, 2015). In closed system aqua-
steinholm, 2012; Blaser & Rosemberg, 2012); illustrating culture, nitrogenous wastes are a particular challenge
the complexity of interactions between lighting, back- because the goal of providing constant feed to larval
ground, and behavior in this species. All of these data fish tends to promote overfeeding and excessive
highlight the sensitivity of the fish to lighting conditions ammonia production in rearing tanks.
and support the idea that this parameter is deserving of Zebrafish adults and larvae appear to be well adapted
further examination. to deal with relatively high levels of environmental
Water flow rates will also impact larval fish perfor- ammonia (Braun, Steele, Ekker, & Perry, 2009; Kumai,
mance. At their earliest stages, most larval fish are faced Harris, Al-Rewashdy, Kwong Raymond, & Perry, 2015;
with the fundamental challenge of effectively maintaining Shih Horng, Hwang, & Lin, 2008). Larval zebrafish
their position within the water column, especially as it have been shown to survive and grow well in static cul-
relates to prey capture and ingestion (China & Holzman, ture tanks where unionized ammonia levels are as high
2014). Effectively, larval fish need to consume enough as 0.16e0.18 parts per million (ppm) (Best et al., 2010).
nutrients to satisfy their demands for both energy This elevated tolerance might be related to the salinity
(required to combat drag) and growth. At the same levels of the water in this particular study (5 parts per
time, the flow rate will also affect prey availability, and 1000 or ppt); in a number of other fish species, ammonia
this can be more or less pronounced depending upon toxicity is reduced when animals are held at elevated
the size and density of the particle. Finally, flow rates salinities (Da Silva, Coimbra, & Wilson, 2009; Sampaio,
will also impact water quality; in general, higher rates 2006). Still, zebrafish larvae display remarkable flexi-
of flow will result in lower accumulations of nitrogenous bility in the way they are able to transport ammonia;
wastes and higher dissolved oxygen. Larval rearing pro- in one study where they were challenged with high
tocols must strike a balance between all of these forces environmental ammonia along with conditions of high
while taking into account the biology of the target alkalinity (pH of 10.0) that inhibit ammonia excretion,
species. they were able to either to elevate their excretion of
Zebrafish larvae are not adapted to high flow condi- ammonia (if not acclimated to the high alkalinity) or
tions. Elevated rates of flow into tanks has been shown convert ammonia to urea (when acclimated) (Kumai
to reduce survival and growth (Bagatto, Pelster, & et al., 2015). This is an excellent demonstration of the
Burggren, 2001). This is, presumably, because the adaptability of the zebrafish to varying conditions, a
increased energy demands associated with maintaining character trait that reflects their evolutionary history in
a position in the water under elevated flow come at the habitats subjected to wide ranges in seasonality and
expense of prey capture and growth, an idea supported environmental change (Arunachalam et al., 2013;
by the fact that performance is poorest in the youngest Engeszer et al., 2007).
fish exposed to the highest rates of flow (Bagatto et al., Zebrafish larvae also display some level of tolerance
2001). This is an important point to consider in the to environmental nitrite. In one study, juvenile fish
development of rearing protocols for this species, espe- from 20 dpf showed significant depression in growth
cially those that rely on higher rates of water exchange rates when exposed to 107 and 130 mg/L NO-2 for
to control chemical waste. 28 days (Voslárová, Pisteková, Svobodová, & Bedá
nová,
2008). Earlier exposures (96 h postfertilization) cause
developmental abnormalities and functional defects at
concentrations as low at 100 mg/L (Simmons, Karimi,
Chemical Talwar, & Simmons, 2012). Trace amounts of up to
The chemical composition of the water profoundly 0.15 mg/L do not inhibit growth or survival during
impacts the physiology, health, and behavior of the the larval stage (Best et al., 2010).
fish that live in it. So it should come as no surprise Nitrate (NO-3), the tertiary product in the nitrogen
that water quality is a major determinant in the success cycle that is found in recirculating aquaculture systems
or failure of any given fish larviculture program. Various (Swann, 1997), is generally far less toxic to larval fish
chemical parameters influence larval performance, but than its precursors (Camargo, Alonso, & Salamanca,
the most proximate of these are nitrogenous waste 2005), and this is certainly the case for zebrafish

III. Husbandry
372 32. Zebrafish Larviculture

(Lawrence, 2007). One comprehensive study recom- McCormick, 2009; Einum & Fleming, 1999; Platenkamp
mends minimal safety values of 1450, 1855, and & Shaw, 1993; Vallin & Nissling, 2000; Vijendravarma,
1075 mg/L for embryonic, newly hatched, and swim- Narasimha, & Kawecki, 2010). There is evidence that
up larvae, respectively (Learmonth & Carvalho, 2015). maternal condition will influence egg size in a number
While zebrafish larvae display tolerance to higher of fish species, including brown trout (Einum & Fleming,
levels of nitrogenous wastes than many other aquacul- 1999), Pacific cod (Vallin & Nissling, 2000) and mosquito-
ture species, the management goal should be to main- fish (O’Dea et al., 2015). This phenomenon is often related
tain total ammonia and nitrite values at or near zero to the nutritional state of the parents; offspring derived
and nitrate values well below the minimal safety values from well-fed parents survived at higher rates than
referenced above (Harper & Lawrence, 2010). This high- offspring from feed-challenged individuals in the marine
lights the importance of making a distinction between fish Acanthochromis polyacanthus150. It may also be related
tolerance and optima. The fish may be adapted to deal to other factors, including the size and age of the mother
with elevated waste levels, but there is always an energy (Kindsvater, Rosenthal, & Alonzo, 2012). Stress has also
tradeoff involved; any energy expended in maintaining been shown to influence egg quality and survival in
homeostasis under varying conditions is energy lost several species (Campbell et al., 1992, 1994; Mccormick,
from growth and overall performance. Certainly, 1998).
managers should look to avoid “challenging” fish with This relationship has been explored to some extent in
sub-optimal conditions; doing so will always result in zebrafish. However, several studies have highlighted the
depressed performance; in some cases, mortality. This association between maternal diet and embryo survival
applies not only to nitrogenous wastes, but also to any and quality. Miller and coauthors showed that zebrafish
other environmental parameter that the developing fed a vitamin E deficient diet produced significantly
fish may experience. more embryos with developmental abnormalities and
Given the prevalence of the zebrafish as a model or- a reduced rate of survival (Miller et al., 2012). In another
ganism, it is perhaps surprising that there are relatively study, zebrafish with access to more food produced
few published studies in the literature on the impacts of offspring that were more likely to survive to adulthood
water chemistry on growth and survival, other than and showed higher levels of physical activity than fish
nitrogenous wastes. The one other environmental that were produced by adults subjected to feed restric-
parameter that has been delineated for zebrafish larvae tion (Newman, Jhinku, Meier, & Horsfield, 2016). These
is salinity. A common rearing practice involves cogrow- data demonstrate how critical environmental conditions
ing zebrafish with rotifers in brackish water at salinity experienced by broodstock are for larval performance.
values of 5 mg/L (Best et al., 2010; Lawrence et al.,
2016) Additional studies on the salinity tolerance of
zebrafish larvae show that their tolerance increases Methodology
with age; 2e4 h old embryos exposed to salinities above
6 mg/L for longer than 1 h displayed significantly Given the plethora of factors that contribute to suc-
elevated mortality versus control, and none survived cess in larval rearing protocols, there is no standard
at 8 mg/L when exposed for 2 h (Sawant, Zhang, & Li, approach to zebrafish larviculture. The fish can be
2001). However, once embryos were beyond 10 h, they reared on a variety of diets, under a wide range of
tolerated 1e2 h pulses of salinities of up to 10 mg/L conditions. Methodology can be broken down into two
before showing any appreciable signs of stress (Sawant different basic approaches: static and recirculating.
et al., 2001). In at least one other study, larval fish There are also approaches that utilize a blend of both.
(yolk-sac and five to seven dpf stages) showed elevated
mortality at salinity values as low as 2 mg/L (Rothen,
Curtis, & Yanong, 2002), so caution should be used Basic Approaches
when adjusting salinities; in general the fish seem to Larval zebrafish are typically grown in either static or
be more tolerant to salt once they inflate their gas blad- recirculating tanks. In static rearing approaches, the fish
ders (Best et al., 2010; Lawrence et al., 2016). are cultured in containers that receive only periodic wa-
ter exchanges, either automatically or manually. In gen-
eral, static approaches should incorporate the use of live
Epigenetic Factors feeds that persist, if not thrive, in these conditions. The
best-known examples of these are ciliates, brachionid ro-
Parental Condition tifers, and cladocerans (e.g., Daphnia). The use of these
types of feeds will minimize waste production and
The concept that the environment experienced by the
decrease the required frequency of water exchange. Pro-
parent, especially the mother, has an influence on the
cessed feeds and live feeds that do not persist in fresh-
phenotype of their offspring is well established in many
water (i.e., Artemia nauplii) tend to break down and
organisms, including fishes (Donelson, Munday, &

III. Husbandry
 Future Directions 373
produce more ammonia, thus necessitating a greater fre- number of studies demonstrate this, most notably one
quency and volume of water exchanges to minimize published by Biga and Goetz that showed that zebrafish
pollution that will limit fish growth. This breakdown fed on Parmecium only for the first 4 weeks of life attained
of organic waste will also consume and therefore limit a total mean length of only w4.7 mm at 28 days postferti-
dissolved oxygen (Swann, 1997). lization (Biga & Goetz, 2006). This compares very unfa-
Many culturing methods for zebrafish employ a vorably with a number of other papers that show that
blend of the two approaches, usually starting with live larval zebrafish fed other diets (Artemia (Conceic, 2010),
diets in static conditions before progressing to processed rotifers (Aoyama et al., 2015; Best et al., 2010; Farias &
diets or some admixture of live and processed feeds in Certal, 2016), or processed feeds (Carvalho et al., 2006;
recirculating or flow-through tanks. The higher protein Hensley & Leung, 2010)) all attain a significantly larger
content and mass of the processed diet or admixture of size at a similar stage of development.
live/processed feeds will necessitate a rate of water In principle, it is possible to grow zebrafish exclu-
exchange that can be most practically achieved through sively on a processed diet from first feeding onward.
a constant exchange to control ammonia and maintain Historically, to achieve this necessitated a sacrifice of
dissolved oxygen at adequate levels. either growth or survival (Goolish & Okutake, 1999),
but recent advances in diet formulation and feeding
practices allow for relatively high values of both metrics
(Farias & Certal, 2016; Hensley & Leung, 2010; Onal &
Published Methods Langdon, 2005; Önal & Langdon, 2016). Interestingly,
There are a number of published methods for rearing the addition of live diets to protocols that utilize pro-
zebrafish larvae in the literature from the past several cessed feeds tends to improve growth and survival
decades. The first and most notable of these appeared (Farias & Certal, 2016).
in the Zebrafish Book68, which described a basic protocol This supports the generally held perception that larval
for rearing “baby” zebrafish centered on the use of first zebrafish, as is the case for many other aquaculture spe-
Paramecium sp. and then Artemia nauplii in static cies, perform best on live diets. Zebrafish larvae fed on
culturing containers before introduction of the larvae Artemia, either exclusively (Carvalho et al., 2006; Conceic,
at 21 dpf into recirculating tanks where they continued 2010), or in progression after rotifers (Lawrence et al.,
to be fed Artemia until they were eventually transitioned 2015), perform very well. The “polyculture method,”
to a flake diet (Westerfield, 2007,). Another variation of where larval zebrafish and rotifers are grown together
this method was published in another popular book, in static, brackish water for the first week of develop-
Zebrafish: A Practical Approach, although this protocol ment, supports some of the highest performance metrics
incorporated the use of processed, powdered diet, rather published in the literature (Best et al., 2010; Lawrence,
than live feeds, in static conditions with zero water ex- Adatto, Best, James, & Maloney, 2012d, 2016). Critically,
change for the first few weeks of life (Nusslein- this particular approach is likely so successful because
Volhard & Dahm, 2002). These two basic approaches, it meets many of the criteria (discussed above) that are
or various derivations of them, have been utilized by critical to developing, first feeding larvae: a simple to
laboratories all around the world to grow zebrafish for digest, nutritious, and attractive prey item that can be
several decades. saturated in the water without polluting it, maximizing
Indeed, the efficacy of these methods for rearing encounter rates for the fish. The rotifer, when presented
larval zebrafish has never been in doubt; the model sys- in this fashion satisfies many of these requirements, but
tem experienced dramatic growth in popularity during other diets and approaches can be adapted to achieve
this period. This “success” can probably be attributed similar results.
to the considerable tolerance of the fishdeven during
the larval stagesdto varying environmental conditions.
However, it is important to note that these methods were Future Directions
never optimized or subjected to rigorous peer review.
Consequently, there is no published performance on sur- The state of zebrafish larviculture has advanced
vival or data associated with these methods, and it is considerably in the past 5 years. Still, there are many
difficult to tell how effective they really are. areas in need of improvement. The first is simple: the
In the past five to 10 years, there has been an increased newest methodologies need to be more widely applied
focus on optimizing larval rearing protocols beyond than they are. A very large proportion of labs still practice
these early approaches. These data highlight a number very basic larval rearing approaches that have remained
of basic generalities about zebrafish larviculture. One is largely unchanged from the primitive methods published
that Paramecium, while it supports good survival of first in the Nusslein-Volhard and Westerfield books (Lidster,
feeding zebrafish, does not promote robust growth. A Readman, Prescott, & Owen, 2017). It is very likely that

III. Husbandry
374 32. Zebrafish Larviculture

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