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169
Comparative Medicine Vol 72 No 3
Copyright 2022 June 2022
by the American Association for Laboratory Animal Science Pages 169–180
Introduction
Betta splendens, also called Siamese ghting sh, or simply
‘betta,’ are anabantoids, a suborder of sh that possesses a
labyrinth organ that allows them to breathe air from the sur-
face of the water.29,55 The Betta genus contains many different
species that are endemic to parts of Southeast Asia. They are
usually found in shallow, still bodies of water, including rice
paddies and areas that ood during the rainy season.54,69 Betta
are a unique potential model for evolutionary, neurobiologic,
and behavioral studies. Although they have been studied sci-
entically for over one hundred years, a lack of standard and
effective husbandry practices has hindered wider adoption of
the species as a model system.
Betta splendens exhibit aggressive territorial behaviors, and
in 18th and 19th century Thailand, pairing 2 opponent sh in
staged ghts became a national pastime.69 By the 19th century,
both wild-caught sh and domesticated sh were used in these
staged ghts. Some written reports suggest domestication be-
gan around 1850,69 but other reports suggest the practice likely
began over 600 y ago.5 Recent genetic analyses are consistent
with domestication beginning at least 400 y ago.41 As a result
of hundreds of years of selective breeding for ghting ability,
domesticated betta are now highly aggressive as compared
with wild betta.76
Variations in color and morphology also arose as a result of
domestication (Figure 1). This diversication, along with im-
portation of betta to Europe and the United States in the early
1900s, led to a boom in popularity. Ornamental strains of betta
can now be purchased in most pet stores, and the wide variety
of different colors, patterns, and n types makes them a popular
sh for aquarium hobbyists. Soon after their introduction into
the aquarium hobby in the early 1900s, a scientic interest in
betta emerged. The earliest studies on betta investigated the
patterns of inheritance of certain morphologic traits,27,42,78
experimented with sex reversal,71 and described behaviors such
as nesting9,11,28,59 and aggression.47,66,72
Here we review betta’s past and present use in scientic
research and detail the effective, streamlined practices we have
developed for keeping and propagating betta in the lab. Our
goal in providing this information is to facilitate the adoption
of betta as a model system, paving the way for future work on
betta husbandry, genetics, development, evolution, behavior,
and neurobiology.
Betta as a Model for Social Behaviors
and Neurobiology
Betta are a unique model for studying complex social behav-
iors. Aggression in betta is highly robust and stereotyped.66 A
male betta, when presented with an opponent male, will are
his ns, erect his gill covers, and perform lateral tail beating
movements. Male sh will engage in this behavior even if they
are in separate tanks. The male aggression response can also be
elicited by allowing the sh to see their reection in a mirror.47
While a number of model organisms are used to study aggres-
sive behaviors (for example, mice and fruit ies), aggression
in those species depends largely on olfactory cues. However,
in betta, visual cues alone are sufcient to evoke an aggressive
response, making them particularly suitable for the study of
visually evoked aggression.
Betta are also useful as a model for paternal behavior. Betta
splendens are bubble nesters; prior to mating, males create a
bubble nest for developing offspring by blowing mucus-coated
bubbles at the surface of water.9 Betta spawning takes an
Original Research
Care and Use of Siamese Fighting Fish
(Betta splendens) for Research
Madison R Lichak,1,2 Joshua R Barber,3 Young Mi Kwon,1,2,4 Kerel X Francis,1,2 Andrés Bendesky1,2,*
Betta splendens, also called Siamese ghting sh or ‘betta,’ are a popular species in the shkeeping hobby. Native to South-
east Asia, betta have been selectively bred for their ghting ability for hundreds of years, which has resulted in the species’
characteristic male aggression. More recently, betta have been bred for a number of ornamental traits such as coloration, n
morphology, and body size. Betta have unique characteristics and an evolutionary history that make them a useful model
for studies in the elds of behavior, endocrinology, neurobiology, genetics, development, and evolution. However, standard
laboratory procedures for raising and keeping these sh are not well established, which has limited their use. Here, we
briey review the past and present use of betta in research, with a focus on their utility in behavioral, neurobiological, and
evolutionary studies. We then describe effective husbandry practices for maintaining betta as a research colony.
Abbreviations: dpf, days post-fertilization.
DOI: 10.30802/AALAS-CM-22-000051
Submitted: 4 Feb 2022. Revision requested: 21 Mar 2022. Accepted: 16 May 2022.
1Zuckerman Mind Brain Behavior Institute, 2Department of Ecology, Evolution and
Environmental Biology, 3Institute of Comparative Medicine, and 4Department of Biological
Sciences, Columbia University, New York, USA.
*Corresponding author. Email: a.bendesky@columbia.edu
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average of 5 h,59 during which eggs are released a few at a time
by the female and are fertilized externally before falling down
the water column. The male retrieves approximately 90% of the
fallen eggs in his mouth,59 with limited help from the female,
and places them into the bubble nest. Eggs and newly hatched
larvae remain in the bubble nest for up to 3 d, and the male
usually stays in the immediate area, picking up fallen eggs or
larvae in his mouth and returning them to the bubble nest.12
Aggressive and paternal behaviors, and factors that modulate
these behaviors, have been characterized in previous laboratory
studies. Studies on betta aggression have examined the effects
of pharmacological interventions on aggressive responses46,73
and how aggression is modulated by the environment,15,31,32
prior social experience,10,14,20 and opponent characteristics.3,8,47
Studies of nesting behavior have revealed the composition
of bubble nests36 and how nest characteristics are altered by
environmental conditions35 and competition for resources.11
This characterization of behavior, combined with an under-
standing of the brain regions involved in aggression13 and the
availability of brain atlases45 provides a strong foundation for
future investigation of the neurobiology and neural circuitry
underlying these behaviors.
Betta as a Genetic Model for the Evolution of
Development and Behavior
The evolutionary history of betta makes this species ideal for
studying the evolution of behavior and morphology, and the
genetic and genomic bases of betta traits. Some of the earliest
studies on betta examined the Mendelian inheritance patterns
underlying certain morphologic traits such as Cambodian
patterning27,78 and blue coloration.42 Previous studies have
also investigated the cellular architecture of pigmentation in
betta,4,37,38 providing additional foundation for genetic analyses
of coloration and patterning.
Annotated reference genomes for wild41 and domesticated22,57,79
betta are now available, enabling powerful genetic and
genomic analyses. The B. splendens genome is only 440–450 Mb,
one of the most compact vertebrate genomes, resulting in
lower costs for sequencing-based studies. Recent work using
genome-wide association studies and quantitative trait analyses
have uncovered candidate genes involved in sex determination,
coloration, and n shape.41,79 Some studies have evaluated these
candidate genes through induced mutations with CRISPR/
Cas9,79 laying the groundwork for the future use of modern
genetic tools to study betta.
Betta splendens, and the Betta genus in general, also have great
potential as models for studying domestication and population
genetics. Over 70 species of Betta are currently recognized, with
new species, such as B. mahachaiensis, being described only re-
cently.40 The mitochondrial genomes of many Betta species have
recently been sequenced,2,56,67 and their genome-wide variation
relative to B. splendens has been characterized,41 providing a
strong basis for future phylogenetic analyses. Recent genomic
analyses have revealed that betta were likely domesticated over
400 y ago.41 Genetic evidence also indicated that the modern
domesticated Betta splendens has hybridized with other species in
the B. splendens species complex: B. imbellis and B. mahachaiensis.41
Future studies could examine the behavioral, morphologic, and
evolutionary consequences of this hybridization.
Housing And Water Parameters
Good water quality is critical for keeping and raising healthy
betta. Like many aquarium species, betta are susceptible to ill-
ness and disease when kept in water of poor quality. Ammonia, a
byproduct of the breakdown of sh waste and uneaten food, has
detrimental effects on health (see “Health and Disease” below)
at levels above 0.25 ppm. To maintain good water quality, we use
a standalone recirculating housing system (Figure 2A) designed
for housing zebrash (Aquaneering, San Diego, CA), in which
water moves continuously through tanks and passes through
multiple ltration steps. In our system, water is mechanically
ltered through a particulate lter mat, which captures large
debris and uneaten food. The water is then directed through
a biologic lter, which breaks down ammonia into less toxic
nitrites and nitrates. In the third ltration step, the water ows
through a set of carbon lters that remove chlorine and organic
compounds. Finally, the water passes through UV light to kill
pathogens. Clean water is then directed into individual tanks
at a rate of approximately 150 mL per minute, displacing water
Figure 1. Six varieties of Betta splendens, varying in n type, color, and patterning. A) blue veiltail male ornamental, B) red crowntail male or-
namental, C) mosaic short halfmoon male ornamental, D) yellow plakat male ornamental, E) plakat male ‘ghter’ strain raised in Thailand for
betta ghting competitions, F) wild-caught male Betta splendens.
171
Care and use of betta sh in the laboratory
from the back of the tanks, where it begins the ltration process.
We replace 15% of the total water volume in our system daily
with fresh reverse osmosis water; this replacement removes
nitrites and nitrates that build up through the biologic ltration
step described above. We use an aquarium water test kit (API
Freshwater Master Test Kit, API/Mars Fishcare North America,
Chalfont, PA) weekly to determine the ammonia, nitrite, and
nitrate levels on our system. These processes reliably maintain
nondetectable ammonia, nitrite, and nitrate levels in hundreds
of tanks on our system.
pH and salinity. Betta can tolerate a pH range63 of 5.0 to 9.0 and
salinity levels81 of up to 9.35 mS/cm (6,000 ppm). Despite the
range of values tolerated by betta, we aim to keep the pH and
salinity of our system stable to minimize potential behavioral
variation in our experiments. Our system is maintained at a pH
of 7.0 (range 6.9 to 7.2), and a salinity of 1.0 mS/cm (range 0.9 to
1.1 mS/cm or 600 to 700 ppm). The addition of small amounts
of salt to freshwater aquaria can reduce disease48,49,58 and
improve sh growth.7 The daily 15% freshwater replacement
introduces reverse osmosis water to the system, which lowers
the salinity and pH. However, we maintain consistent pH and
salinity levels through an automatic dosing system that adds,
dropwise, a solution of sodium bicarbonate (65 g per 3 L water)
or aquarium salt (Instant Ocean, Blacksburg, VA; 280 g per 3 L
water; for composition see ref 34) until the desired pH or salinity
is achieved. If it is necessary to reduce the pH or salinity of the
system, we add more fresh reverse osmosis water.
Water temperature and light cycle. Our system water tempera-
ture is kept at 28 °C, which is ideal for bubble nest construction33
and ovarian development65 in betta, and is consistent with
common betta husbandry practices.60,77 Furthermore, growth
at lower temperatures (23 to 25 °C) might lead to a biased sex
ratio with a high incidence of females,80 whereas growth at
higher temperatures (33 ºC) increases larval mortality and may
lead to a higher incidence of males.24 At 28 ºC the sex ratio is
usually about 50% of each sex. The 28 ºC system temperature
also reduces the viability of some pathogens,50 aiding in disease
prevention. We obtain a consistent water temperature of 28 °C
by setting the room to 26 to 27 °C (the highest temperature that
central heating in our building can provide) and using water
heaters in the sumps of our housing system to supply additional
heat. Our colony is maintained on a 14:10-hr light-dark cycle
(using uorescent bulbs for illumination), which mimics sum-
mer lighting conditions in betta’s natural geographic range.
Published studies have not determined the optimal lighting
conditions for betta reproduction. Our lighting conditions
are based on the assumption that betta reproduce best during
Thailand’s rainy season, which coincides with the summer
months. Therefore, we use light-dark cycles that occur during
the summer months.
Housing. If good water quality is maintained, adult betta
can be effectively housed in a variety of tank sizes.64 In our
colony, adult sh are individually housed in 0.8-L or 1.4-L tanks
(Figure 2B) on our recirculating system. Due to the aggressive
Figure 2. Betta housing system. A) Standalone recirculating housing system, with automated dosing system and rows supporting multiple
individual tanks. B) Top: 2.8-L tanks housing multiple late larval sh; bottom: 1.4-L tanks housing single adult sh. White acrylic dividers are
placed between tanks holding adult sh to prevent aggressive display. Individual tubing provides constant water ow to each tank, displacing
water from the back of the tank where it begins the water ltration process. C) Side view of 1.4-L tank containing a single adult sh and suction
cup plant enrichment item. Lids and bafes at the back of the tank prevent sh from escaping into the system.
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behavior of male betta, adult males are individually housed.
We place opaque, white, nonreective, plastic dividers be-
tween tanks containing males to prevent aggressive display
in response to neighboring sh. We also tend to individually
house adult female betta, but we have had success cohousing
them at a density of 2 sh per L, provided these tanks are
carefully monitored for excessive aggression. If particularly
aggressive females are identied in tanks with cohousing, the
aggressive sh are removed and individually housed. We also
cohouse betta while rearing larvae and juveniles, as described
in further detail in the “Feeding and Rearing” section of this
manuscript.
Wild betta and other strains tend to jump through the holes on
the lid used to introduce food, or through small gaps between
the lid and the tank. Therefore, lids must always be properly
secured, and feeding holes in the tank lids can be covered using
mesh tape (Duck Brand, product #282084, Avon, OH). The holes
of the mesh tape are about 3 mm wide and are convenient for
feeding, but are too small for sh to go through.
A build-up of algae may occur on tank walls after a few
months, impeding the view into the tank. When this occurs,
sh should be transferred to a clean tank. Aquaneering tanks
are made of polycarbonate and can be washed in a standard
facility tunnel or rack cage wash machine at 83 °C without de-
tergents. We do not recommend autoclaving the tanks as our
attempts have caused warping and reduced the transparency
of the plastic.
Enrichment. Environmental enrichment may reduce abnormal
sh behaviors,23 and studies have shown that enriched areas are
preferred by tank inhabitants.70 For enrichment purposes, we
provide each tank with one of 2 types of plastic plants (Imagi-
tarium, a Petco brand, San Diego, CA). The rst type of plant
adheres to the side of the tank with a suction cup (Figure 2C).
When this type of plant is placed within a few centimeters of
the surface of the water, we often observe sh lying on this plant
like a hammock. The second type of plant enrichment oats on
the surface and hangs down into the water. Plant enrichment
items should be carefully checked to assure that they are not
impeding water ow in the tanks. Before introducing new plants
to tanks, or when they become visibly dirty, we scrub them clean
of organic materials and soak in a 5% bleach solution for 24 h
before carefully dechlorinating them in a solution of 10 g/L
sodium thiosulfate.
Breeding and Reproduction
Methods for setting up betta mating tanks vary widely in
the hobbyist and commercial communities. We have had suc-
cess using 5-L acrylic tanks (30 cm × 12 cm × 22 cm high) for
mating (Figure 3A). We cover 3 sides of the tank with brown
paper to prevent the breeding pairs from seeing into adjacent
tanks or being disturbed by movement in our sh room. The
mating tanks are lled with system water to a height of ap-
proximately 10 cm and are static (that is, they are not connected
to our recirculating system). We place at heat mats (VivoSun,
Figure 3. Betta mating set up. A) 5-L static mating tank containing plants and stones for sh to hide. B) Male ornamental betta with bubble nest
built under Indian almond leaf. C) Nongravid female betta. D) Gravid female betta; note the rounded belly (arrow).
173
Care and use of betta sh in the laboratory
Ontario, CA), designed for use under potted plants or reptile
tanks below the tanks to keep the water at 28 °C. Because Betta
splendens are bubble nesters, we provide each tank with a oat-
ing substrate under which the male can build his nest. Hobby
and research breeders use various materials for this purpose,
such as small pieces of bubble wrap, Styrofoam cups cut in half
lengthwise, and plastic container lids. We use a piece of dried
Indian almond leaf (Terminalia catappa from SunGrow through
Amazon.com; Figure 3B) as the bubble nest substrate. Indian
almond leaves release tannins into the water, which slightly
reduce the pH and may also decrease the risk of disease.43
Regardless of the potential health benets, we have found
that it is easier to harvest eggs from bubble nests built under
Indian almond leaves than from bubble nests built under other
substrates. We also add items in each tank to provide places
in which sh can hide from aggressive, persistent partners
(Figure 3A). Plastic cylinders, terracotta pots, and either silk
or live plants can all provide shelter. Finally, we place acrylic
covers over all tanks to maintain high levels of humidity (90%
humidity in mating tanks, compared with approximately 25%
in the sh room), which helps to keep the bubble nest intact.12
Multiple methods can be used to introduce breeding pairs
to the mating tanks. Many breeders start by placing the female
behind a clear divider, such as inside a plastic cup, which allows
the pair to see each other, but not physically interact; this can also
allow the male to build a bubble nest without interruption. In
this method, the divider is removed the next morning, giving the
female access to the male. However, we have found that sepa-
rating the pair is not necessary for successful mating; doing so
increases the work required without improving mating success
rates or reducing female injuries. However, variations on this
method could be useful under certain circumstances, such as for
observing mating behaviors or when precise timing of mating
is important (for example, when planning to inject zygotes for
genetic manipulation). In our routine mating method, we place
the male and female together in the tank without a divider. We
usually add mating pairs to the tanks 3 to 4 h before the lights
are set to turn off and watch for mating the following morn-
ing. With these methods, we achieve an overall mating success
rate (dened as the presence of eggs within the maximal 3-d
period during which the pair is in the mating tank) of approxi-
mately 50% for all betta varieties (ornamentals, wild-caught,
and ghters), consistent with success rates obtained with other
laboratory methods.17
Most pairs begin mating between 1 and 5 h after light onset,
and approximately 90% of our successful matings occur the day
after introduction. During the mating process, the female will
release a few eggs during each ‘embrace’ with the male, who
fertilizes eggs every minute or so over the course of 1 to 4 h as
they are being released. Mating is complete when the female
retreats, often to a part of the tank where she can hide from the
male. As soon as pairs nish mating, we remove the female and
male with a net and place them back in their home tanks before
collecting eggs from the bubble nest to be raised in vitro. The
number of eggs released during mating ranges from 12 to 492,
with a mean of 252 eggs (Figure 4A). This variation could be
due, at least in part, to factors such as age, inbreeding, strain,
and recency of previous matings.
With our routine mating method, serious mating-related
injuries are rare. While minor nipping and biting is expected,
sh should be observed after introduction to ensure that neither
individual is excessively aggressive. If either individual expe-
riences sustained biting, the mating pair should be separated,
and mating reattempted at a later date. However, we have also
found that some sh are repeatedly very aggressive during
mating, regardless of whether we use our routine mating setup
or the divider method; these sh may be unsuitable for mat-
ing. If sh sustain minor injuries during mating, we prevent
infection by using a prophylactic 5-d course of methylene blue
(Methylene Blue, Kordon, Hayward, CA), which has antifungal
properties.16,74 We add methylene blue daily to the home tanks
of injured sh at a nal concentration of 3 ppm (0.0003%), and
we remove these sh from the recirculating system during this
treatment.
In our experience, a number of variables contribute to the
successful mating of a breeding pair. Females that appear gravid
(Figure 3C, D) and males that build bubble nests in their home
tanks make ideal breeders, although the presence of either of
these characteristics is not necessary for sh to successfully
mate. Undernourished and underfed sh rarely mate success-
fully. When we tried feeding our adult colony only dried food,
the mating success rate fell to around 30%, an observation
backed up by the literature.6,44 Therefore, we feed our colony
live, newly hatched brine shrimp (for more information on feed-
ing protocols, see “Feeding and Rearing” below) at least once
per day. In addition, we have not observed successful mating
in any pair kept in a mating tank for longer than 3 d.
Figure 4. Clutch sizes and mating success. A) Distribution of clutch
sizes. Black line at mean clutch size = 252 eggs (range 12 to 492 eggs;
n = 79 matings). B) Age of sh of each sex at breeding according to
outcome of mating (“success” means animals mate and release eggs).
Comparisons were performed using a 2-sided Mann-Whitney test
with Bonferroni correction (successful n = 56, unsuccessful n = 62; *,
P ≤ 0.05).
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Age and the timing of previous matings are also important
factors in mating success. In our experience, females require at
least 4 wk between matings. Females that are mated within a
shorter interval may not release any eggs, or will release eggs
that are nonviable. Males can mate more frequently. We have
had success after waiting only a week between matings of males,
but we have not tried mating individual males more frequently
than 2 wk in a row. Successful breeders tend to be younger, and
we have not seen successful mating with sh older than 40 wk
(Figure 4B) (the life span of betta is around 2 y under laboratory
conditions). Fish raised under our conditions can successfully
mate as early as 10 wk after fertilization if individually housed
for at least 5 d starting at 9 wk after fertilization.
Feeding and Rearing
Hatchling stage (0 to 4 dpf). We routinely raise developing
embryos and newly hatched larvae from 0 to 4 d post-fertiliza-
tion (dpf) in glass culture dishes; this allows us to monitor the
number of eggs released and embryo survival rates. The chorion
of developing betta sticks easily to plastic, so we use glass tools
when handling eggs and unhatched, developing sh. Eggs are
removed from the bubble nest shortly after mating, either by
inverting the bubble nest substrate directly into a glass culture
dish, or by using a spoon to scrape the eggs from the bubble
nest. Once collected, the eggs are maintained in a glass culture
dish containing freshly made E3 medium (5 mM NaCl, 0.17 mM
KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 0.0001% methylene blue)
for 5 d. E3 contains methylene blue, which limits bacterial and
fungal growth.26,39 Culture dishes containing developing sh
are kept in an incubator (Heratherm, Thermo Scientic) set at
28 °C. Our incubator has a glass door that allows the entry of
ambient light from our main laboratory space. This lab space
is lighted for roughly 12 h per day (from around 7:00-19:00)
with white and yellow LEDs, and has windows to the outside
of the building. Larval sh begin hatching between 29 and 44
h after fertilization19,30,75 and begin swimming by 72 h after
fertilization.30 We use a glass transfer pipet daily to remove
unfertilized eggs or embryos that are not developing or that
are clearly malformed. Daily removal of nonviable embryos is
necessary to prevent pathogen growth and an increase in am-
monia. Yolk reserves are maintained in developing embryos
and newly hatched larvae through 120 h after fertilization,30 so
we do not feed sh while they are in culture dishes (Figure 5).
Early larval stage (5 to 14 dpf). At 5 dpf, larvae are moved from
culture dishes to 2.8-L tanks that are lled with 1 L of system
water (that is, to a water height of about 5 cm). Because larval
sh at this stage are very small (from approximately 2 mm at
5 dpf to 8 mm at 14 dpf), they are kept in relatively shallow,
still water that gives them easy access to the surface. Access to
the surface of the water prevents hypoxic conditions that are
associated with reduced survival.6 In the age range of 5 to 14
dpf, the sh are too small to be kept on the recirculating system
without being washed out of the tanks, and housing larval sh
in tanks with static water allows food to remain in the tanks
for longer periods of time. Each tank is stocked at a density of
approximately 30 larvae per tank (range 20 to 50). Larval sh
can be carefully transferred to these tanks using a glass transfer
pipet, as they will stick to nets and mesh sieves.
On days 5 to 14 after fertilization, we feed the larvae with
Brachionus rotundiformis rotifers. Rotifers have been shown to
increase early betta growth rates as compared with other live
foods.53 We start to feed rotifers at 5 dpf, rather than 3 dpf,
because there is no difference between these two start times in
the size of the sh when they reach 15 dpf (Figure 6). However,
larval size at 15 dpf appears to be lower if rotifer feeding starts
at 6 dpf. Furthermore, starting rotifer feedings on 5 dpf yields
Figure 5. Feeding and housing regimens for in-house raised sh of different developmental stages.
175
Care and use of betta sh in the laboratory
survival rates of approximately 90% of larval sh at 15 dpf, as
compared with approximately 75% survival rate when feeding
begins on 6 dpf.
Rotifers can be cultured easily using a culture bucket, a heater
to maintain temperature at 31 °C, lter media, an air pump, and
a peristaltic dosing machine to feed the rotifers suspended algae
at a rate of 1 mL/hour (culture implements from Reed Maricul-
ture, Campbell, CA). To feed, we harvest 4 L of our 15-L culture
daily (27%) and condense these 4 L down to 250 mL by straining
through a 41-μm sieve and resuspending in system water. As
a cautionary note, if rotifers are being cultured alongside other
food sources like brine shrimp, harvesting equipment for each
culture should be kept separate and cleaned frequently, using
bleach followed by sodium thiosulfate. Contamination of the
rotifer culture with brine shrimp cysts can result in rogue brine
shrimp hatching in the rotifer culture, and these individuals can
consume enough rotifers to cause the culture to crash.
Larval sh are fed with 25 mL of the concentrated rotifer
solution once daily. Well-established cultures have 200 to 350
rotifers per mL (before concentrating) and require little upkeep;
our 4-L harvest gives us enough rotifers to feed up to 10 tanks
of larval sh every day. Some rotifers survive in betta system
water for at least 24 h, giving the larval sh access to food for
the whole day. Ammonia levels in larval tanks receiving daily
rotifers remain at 0 to 0.25 ppm from 5 to 14 dpf, so we do not
perform any water changes during this period.
In developing our maintenance protocol, we experimented
with feeding paramecium, which we found to be more difcult
and time-consuming to culture than rotifers, and with dry
powder food, which rapidly decreased water quality. We also
tried feeding brine shrimp (Artemia nauplii) starting at 5 dpf,
but found that because brine shrimp do not survive for more
than an hour in system water, uneaten dead brine shrimp ac-
cumulated in the tanks. This accumulation led to high ammonia
levels, which required frequent water changes and reduced
larvae survival.
We achieve the best growth rates when early larval sh are
moved to tanks at 5 dpf (that is, raised in culture dishes from
0 to 4 dpf, and then moved to tanks at 5 dpf; Figure 6), but
they can also be maintained in culture dishes through 15 dpf.
Keeping larval sh in culture dishes can be useful for some
experiments. If we raise early larval stage sh in culture dishes,
we keep them in the 28 °C incubator during this time period,
and feed rotifers daily at the same rate of 1 mL concentrated
rotifer suspension per 40 mL water volume. Care must be taken
to diligently remove dead sh and other debris using a transfer
pipet to avoid poor water quality.
Late larval stage (15 to 30 dpf). At 15 dpf, we add a 400-μm
diameter mesh fry screen to each 2.8-L tank and move tanks to
the recirculating system with a slow ow (100 mL per min) of
water (Figure 2B). At this stage, larvae are fed newly hatched
brine shrimp (Artemia nauplii) 2 to 4 times daily, consistent
with feeding protocols in published betta literature.60 Brine
shrimp are cultured daily in a 19-L acrylic hatching cone
(Pentair, Cary, NC) at 28 °C in 8 L of water with a salinity of
25 to 35 ppt, prepared with Instant Ocean aquarium salt. The
culture must be well aerated with an air pump. We start a new
culture each morning and harvest hatched brine shrimp 24 h
later. The correct salinity and temperature are necessary for a
successful brine shrimp hatch; with our methods, suboptimal
hatching rates can almost always be traced to a malfunctioning
water heater in the culture cone or to water salinity outside
the specied range.
Brine shrimp cysts come in 2 varieties: intact (capsulated) and
decapsulated. We highly recommend the use of intact cysts, as
we have found that cultures started using decapsulated cysts
often fail to hatch. We have never had a failed hatch using
intact cysts. When using intact cysts to culture brine shrimp,
care should be taken to remove the capsules before feeding the
shrimp to the betta. Betta will eat the capsules, which leads to
gut blockages and buoyancy issues. Capsules can be separated
from hatched shrimp by stopping aeration and allowing the
capsules to oat to the surface, before harvesting the hatched
shrimp from the lower portion of the culture cone using the
spout at the bottom. We purchase shrimp with intact cysts that
have been coated in a nontoxic layer of iron (Artemia Interna-
tional, Fairview, TX), allowing easy removal of the capsules
with a strong magnet.
Each tank containing late larval stage sh receives an amount
of brine shrimp that can be consumed between 5 and 15 min
(that is, approximately 20 to 50 shrimp per sh, depending on
age). If larvae are overfed, uneaten brine shrimp may form a
Figure 6. Betta size at 15 dpf under different raising conditions. All sh were raised in culture dishes in a 28 °C incubator from 0–4 dpf, then
either moved to 2.8-L tanks for 5–15 dpf (orange points) or kept in culture dishes for 5–15 dpf (blue points). Daily feeding with rotifers for both
housing conditions began on either day 3, 4, 5, or 6 (n≈24 individuals per treatment group). Between housing condition comparisons were
performed using a 2-sided Mann-Whitney-Wilcoxon test with Bonferroni correction. Between feeding condition comparisons for tank-raised
sh were performed using a Kruskal Wallis test, with posthoc analysis performed using Dunn’s test. Nonsignicant comparisons not marked.
(‡, P ≤ 0.001; §, P ≤ 0.0001)
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layer on the bottom of the tank. On the recirculating system,
we have not noticed any health issues or increases in ammonia
caused by a buildup of uneaten food, but this layer can impede
the view into the tank. We move sh to a clean tank if this occurs.
Juvenile stage (31 to 62 dpf). A previous study showed that
juvenile sh raised individually in small water volumes (150
mL) exhibit the most rapid growth.64 However, that study also
found that ammonia levels were highest in the tanks with the
smallest water volume. To keep ammonia levels low while
housing more sh, we raise our juvenile sh in 35-L grow-out
tanks. We move sh to these tanks around 30 dpf, or once sh
reach approximately 1 cm in length. We keep sh at a density of
0.5 to 1 sh per L in these tanks, which is similar to the practices
we observed in betta farms in Thailand. Stocking at a higher
density leads to slower growth. Juveniles in our grow-out
tanks are fed newly hatched brine shrimp 2 to 4 times daily,
and tanks are fed an amount of shrimp that can be consumed
within 5 to 15 min.
Our grow-out tanks contain rocks, terracotta pots, and silk
and/or live plants (Figure 7). Live plants reduce ammonia and
nitrite levels in the tanks, and plants and rocks provide places
for the sh to hide. Each tank has a custom-cut lid to prevent
excessive evaporation that can lead to increased salinity. These
tanks are also hooked up to our recirculating system, with a
water ow that replaces 100% of the water daily, eliminating
the need for time-consuming daily manual water changes. To
remove debris from the bottom of the grow-out tank, we siphon
the bottom of each tank at least once per week. For tanks contain-
ing sh under 2 cm long, we add a piece of mesh to the end of
our siphon to avoid accidentally siphoning up any tank inhabit-
ants. Algae growth is normal in grow-out tanks. Excess algae
can be removed by scraping the sides of the tank with a razor
blade, taking care not to damage silicone sealant in the corners.
Adult stage (63+ dpf). Under our growing conditions, orna-
mental betta can reach sexual maturity by 10 wk of age, which
is 6 to 7 wk faster than is achieved with other methods.62 Fish
in our grow-out tanks begin blowing bubble nests by 8 wk after
fertilization, and around this time some sh can be sexed based
on observation of a female ovipositor. Beginning at 8 wk, we
individually house the largest sh and those that we can sex.
To do this, we move individuals from their grow-out tank to a
0.8-L or a 1.4-L tank and add this tank to the main recirculating
system. Adults placed on the system can breed 5 to 7 d after
removal from the grow-out tanks. Adults on the recirculating
system receive a mixed diet of newly hatched brine shrimp once
daily and food pellets once daily (approximately 10 Golden
Pearls 500- to 800-μm pellets [protein 54%, crude fat 9%, ber
2%, moisture 9%] or 2 to 3 Ken’s 1-mm sh pellets [protein
47%, crude fat 17%, ber 3%, moisture 8%; both from Ken’s
Fish, Taunton, MA), consistent with published betta feeding
protocols.60
Fish cohoused from birth can generally continue to be housed
together indenitely, regardless of their sex, but special care
should be taken to monitor for particularly aggressive sh,
which should be individually housed. We individually house
adult sh on the recirculating system prior to use in breeding
or behavioral experiments, or if they become too aggressive
for the grow-out tanks. Once male sh have been individually
housed, they cannot be reintroduced into a cohoused tank due
to their high aggression.
Health and Disease
Unlike more widely used sh such as zebrash and medaka,
no laboratory strains or pathogen-free sources of betta are cur-
rently available. In addition, to study the wide variety of traits
and genetics present in betta, we often import sh from the
aquarium trade or hobbyist breeders into our colony, and betta
in our colony may be exposed to diseases from these sources. To
reduce the likelihood of pathogens spreading to our colony from
outside sources, we quarantine all new arrivals for at least one
week, with an additional week of quarantine for wild-caught
sh. During the quarantine period, we house sh in static
tanks with an added prophylactic dose of methylene blue (3
ppm, 5-d treatment course) and perform daily water changes
by hand, replacing approximately 100% of the tank water with
fresh water at system pH, salinity, and temperature. We visu-
ally inspect all individuals in quarantine daily for any signs of
infection or illness. While our quarantine practice involves static
tanks and hand water changes, the ideal quarantine program
for betta has not yet been empirically determined, and others
may choose to provide a separate recirculating system to house
their quarantined sh. With that approach, sentinel sh could
be used to alert staff to disease outbreaks.
While a quarantine procedure reduces the number of disease
outbreaks,61 importing sh from multiple sources means that
some pathogens will inevitably enter the system. Brine shrimp
can also be a vector for the transmission of marine bacteria.18
Maintaining pristine water quality is crucial for preventing
Figure 7. Grow-out tanks for housing juvenile and adult betta raised in-house. Grow-out tanks are 35 L, connected to the recirculating system,
contain numerous hiding places, and are stocked at 0.5–1 sh/L.
177
Care and use of betta sh in the laboratory
disease spread,1,21,68 as is recognizing and quickly isolating
sick individuals. We perform a daily health check on all sh,
carefully checking each tank for signs of disease. Poor health
usually leads to recognizable changes in sh behavior. Betta
that are ill often present with clamped ns (Figure 8A), lethargy,
and disinterest in food. Any sh exhibiting signs of disease are
removed from the recirculating system immediately to prevent
spread to other tanks.
Common betta diseases are velvet, n rot, Mycobacterium
infection, and buoyancy disorders. Velvet is a disease caused
by dinoagellate parasites of the genus Oodinium.52 The easiest
way to identify betta with velvet is by observing a coating that
looks like gold dust on the sh’s body. Betta with velvet also
usually have clamped ns, are lethargic, and may stop eating.
If the condition is caught early, we have successfully treated
velvet in our facility using a 5-d treatment course of Proform
C (Pentair, Cary, NC) at a dose of 30 μL per L of tank volume,
with daily 100% water changes. Our only experience with velvet
occurred before the installation of our recirculating system. In
the 3 y after installing our recirculating system, we have seen
no cases of velvet in our colony.
Fin rot is a nonspecic term for lesions on ns caused by either
bacteria or fungi.52 Betta with n rot often have ns with jagged
edges, and the ns may become more transparent and duller in
color (Figure 8B). We have had some success treating mild cases
of n rot with a 4-d treatment course of the antibiotic Furan 2
(nitrofurazone) antibiotic51 at 46 mg/L, with daily 100% water
changes. While rare in young individually housed sh on our
recirculating system, we carefully monitor for this disease in
grow-out tanks. If sh are nipped excessively by tank mates, n
wounds can become infected and n rot can occur. Anecdotally,
sh older than 1.5 y seem to be more susceptible.
Betta can also be infected with Mycobacteria spp. Clinical
signs are nonspecic and can be difcult to distinguish from
other illnesses. Wasting of ns, dull coloration, inactivity
and clamped ns can all be signs of mycobacteriosis. Fish
with clinical signs that cannot be linked to another disease
may indicate a mycobacteriosis outbreak. In these instances,
we euthanize 1 to 2 individual sh and send tissue samples
to an animal diagnostic lab for evaluation for infection with
Mycobacteria. Diagnosis is performed using Ziehl-Neelsen
staining, culture, and/or PCR. All sh with potential myco-
bacteriosis should be isolated to prevent spread to other tanks.
Few rigorous clinical studies have been performed regarding
this condition,25 and we have had minimal success with at-
tempted treatments.
Buoyancy disorders are another health issue we have experi-
enced in our colony. Fish may either oat toward the surface of
the water, or their tails may sink to the bottom so that they swim
vertically in the water. Sinking is more common in developing
sh and is often linked to excessive feeding. As long as the
sh can reach the surface to breathe, sinking issues in grow-
out tanks resolve as sh grow. In our experience, oating sh
show moderate loss of buoyancy control and a round, bloated
appearance of the abdomen. Approximately half of these cases
will resolve with a 24- or 48-h fast, and we characterize these
cases as ‘bloat.’ Some sh seem to be particularly susceptible to
chronic bloat and will be treated multiple times throughout their
life. The other half of our cases of oating manifest similarly
to bloat but often with milder loss of buoyancy and these sh
do not recover after a 48-h fast. Different etiologies are likely to
underlie a bloated appearance in betta sh. Those that recover
may have ingested too much air during feeding, while those
that do not recover may have organ damage associated with
bacterial infection.18
Conclusion
While the use of Betta splendens in the laboratory is currently
limited, their use presents an opportunity for researchers in a
variety of scientic elds. The husbandry protocols we devel-
oped have allowed us to successfully introduce and maintain
thousands of sh in our colony, and we have achieved consistent
mating success, high levels of growth and survival throughout
development, and widespread prevention of illness. We have
accelerated the generation time of betta to 10 wk from a common
standard of at least 16 wk, facilitating genetic tractability. By
following the procedures outlined here, researchers interested
in using betta in their laboratories will be able to maintain a
thriving betta colony under standard conditions that allow them
to generate and raise the large cohorts required for genetic and
genomic analyses, and to perform studies of betta development,
neurobiology, and behavior.
Acknowledgments
We thank Sonia Thomas, Christol Pollard, and the entire Zuckerman
Institute animal husbandry and veterinary staff. We would also like
to thank the many members of the betta breeding community includ-
ing Salvador Alemany, Leo Buss, Gerald Grifn, Liz Hahn, Sieg Illig,
Heidi Mae Burkle, Karen MacAuley, Aurelia Ogles, Holly Rutan, and
Natthacha Frank Sriboribun for providing sh and sharing their
shkeeping expertise. We also thank Peter Lichtenthal for assistance in
collecting early larval growth data. Animal experimentation protocols
were approved by the Columbia University Institutional Animal Care
and Use Committee (AC-AAAT1482). Funding: Searle Scholarship,
Sloan Foundation Fellowship, and National Institutes of Health grants
R34NS116734 and R35GM143051 to AB.
Figure 8. Common signs of diseased betta. A) A sh with clamped ns, a nonspecic symptom that is present in many betta illnesses. B) Adult
male sh with n rot. Note the ‘wasted,’ jagged ns, and the pink necrosis present on the dorsal n.
178
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June 2022
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