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Care and Use of Siamese Fighting Fish (Betta Splendens) for Research


Abstract and Figures

Betta splendens, also called Siamese fighting fish or 'betta,' are a popular species in the fishkeeping hobby. Native to Southeast Asia, betta have been selectively bred for their fighting 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, fin morphology, and body size. Betta have unique characteristics and an evolutionary history that make them a useful model for studies in the fields of behavior, endocrinology, neurobiology, genetics, development, and evolution. However, standard laboratory procedures for raising and keeping these fish are not well established, which has limited their use. Here, we briefly 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.
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Comparative Medicine Vol 72 No 3
Copyright 2022 June 2022
by the American Association for Laboratory Animal Science Pages 169–180
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-
entically 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 diversication, 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 scientic 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 scientic
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 reection 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 sufcient 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
briey 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:
Vol 72 No 3
Comparative Medicine
June 2022
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 zebrash (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.
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 bafes at the back of the tank prevent sh from escaping into the system.
Vol 72 No 3
Comparative Medicine
June 2022
behavior of male betta, adult males are individually housed.
We place opaque, white, nonreective, 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 identied 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
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).
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; 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 benets, 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 (dened 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
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).
Vol 72 No 3
Comparative Medicine
June 2022
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 Scientic) 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.
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 difcult
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 specied 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 (n24 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. Nonsignicant comparisons not marked.
(‡, P 0.001; §, P 0.0001)
Vol 72 No 3
Comparative Medicine
June 2022
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
Fish cohoused from birth can generally continue to be housed
together indenitely, 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 zebrash 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.
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 dinoagellate 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 nonspecic 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 nonspecic and can be difcult 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
While the use of Betta splendens in the laboratory is currently
limited, their use presents an opportunity for researchers in a
variety of scientic 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.
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 Grifn, 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 nonspecic 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.
Vol 72 No 3
Comparative Medicine
June 2022
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... In addition to biological conditions, the predatory efficiency of larvivorous fish is affected by habitat environmental conditions including temperature and acidity (pH). The optimum temperature for larvivorous fish varies, namely 24-28 ºC for P. reticulata and B. splendens, and 24-30 ºC for A. panchax, and with a pH range between 6-7 (9)(10)(11)(12). The ability of larvivorous fish will slow down at the temperature and pH ranges that are not suitable for the optimum conditions due to physiological disorders. ...
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Background: Reduction of the Aedes aegypti population is the priority effort to control dengue virus transmission in­cluding the use of larvivorous fish. Biologically, the predatory efficiency of fish will slow down when the water acidity and temperature change from normal conditions. This study aimed to determine the predatory efficiency of three species of larvivorous fish against the Ae. aegypti larvae in different water temperatures. Methods: Three well-known species of larvivorous fish namely Poecilia reticulata, Betta splendens, and Aplocheilus panchax were placed into 12 cm diameter jars with three water temperature ranges namely 20–21 ºC, 27–28 ºC, and 34–35 ºC, and allowed to three days acclimatization. As many as one hundred 4th-instars larvae of Ae. aegypti were gradual­ly entered into each jar, and a longitudinal observation was made at 5, 10, 30, 60, 120, 240, 360, 480, 600, and 720 minutes. The predated larvae were recorded. Results: In normal temperature ranges, the predatory efficiency of the larvivorous fish was 75%, 72.3%, and 32.8% for B. splendens. Aplocheilus panchax, and P. reticulata, respectively. The predation abilities decreased due to temperature changes. Betta splendens and A. panchax indicated the best predatory efficiency against Ae. aegypti larvae in different temperature conditions. Conclusion: Betta splendens is the best larvivorous fish in the lower to normal, but A. panchax is the best in the normal to higher temperature ranges. This finding should be considered by public health workers in selecting larvivorous fish to control the Dengue vectors.
... In order to facilitate the further expansion of Betta splendens as a model for integrated studies of behavior, cognition, and neuroscience [69], we included a more detailed analysis of our behavioral data. To showcase the depth of data analysis that can be conducted on behavioral data, we incorporated an analysis of behavior and location over time within each assay, similar to an analysis conducted by Ramos et al. (2021) [41]. ...
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The fighting fish Betta splendens, long studied for their aggressive territorial competitions, has the potential to be a tractable and relevant model for studying the intersection of cognitive ecology and social neuroscience. Yet, few studies have comprehensively assessed Betta behavior across both social and nonsocial contexts. Furthermore, the present study is the first to quantify the expression of phosphorylated ribosomal protein S6 (PS6), a proxy for neural response, in the Betta telencephalon. Here, we assessed male Betta behavior across a suite of tasks and found that response to a mirror, but not neophilia (a novel object) nor anxiety (scototaxis), predicted behavior in a social competition. To then explore the cognitive aspects of social competition, we exposed Betta to either a familiar or novel opponent and compared their competitive behavior as well as their neural responses in the teleost homologs of the hippocampus, basolateral amygdala, and lateral septum. We did not detect any differences between familiar-exposed and novel-exposed individuals, but by implementing the first use of a habituation–dishabituation competition design in a study of Betta, we were able to observe remarkable consistency in competitive outcomes across repeated exposures. Taken together, the present study lays the groundwork for expanding the use of Betta to explore integrative and multidimensional questions of social cognition.
... Establishing genetic tools in betta will advance their use as a powerful experimental system to study developmental processes and behavioral traits. Genetic manipulation of betta is facilitated by their reproductive biology: betta fertilize externally and produce clutches of 250 eggs, each with a relatively large diameter of 1 mm (Valentin et al., 2015;Lichak et al., 2022). This enables the microinjection of zygotes in a similar manner to methods for genetic manipulation of zebrafish (Danio rerio) and medaka (Oryzias latipes). ...
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Betta splendens, also known as Siamese fighting fish or “betta,” is a freshwater fish species renowned for its astonishing morphological diversity and extreme aggressive behavior. Despite recent advances in our understanding of the genetics and neurobiology of betta, the lack of tools to manipulate their genome has hindered progress at functional and mechanistic levels. In this study, we outline the use of three genetic manipulation technologies, which we have optimized for use in betta: CRISPR/Cas9-mediated knockout, CRISPR/Cas9-mediated knockin, and Tol2-mediated transgenesis. We knocked out three genes: alkal2l, bco1l, and mitfa, and analyzed their effects on viability and pigmentation. Furthermore, we knocked in a fluorescent protein into the mitfa locus, a proof-of-principle experiment of this powerful technology in betta. Finally, we used Tol2-mediated transgenesis to create fish with ubiquitous expression of GFP, and then developed a bicistronic plasmid with heart-specific expression of a red fluorescent protein to serve as a visible marker of successful transgenesis. Our work highlights the potential for the genetic manipulation of betta, providing valuable resources for the effective use of genetic tools in this animal model.
... The rapid expansion of the ornamental fish industry, together with variable culture conditions, has contributed to a remarkable increase in the prevalence of various diseases (Purivirojkul and Sumontha, 2013;Senapin et al., 2014;Goldstein, 2015;Maceda-Veiga and Cable, 2019), which could also have negative impacts on the development of beta fish farming. Among the numerous diseases affecting betta fish, mycobacterial infections have emerged as a particular problem leading to potential economic impacts (Pleeging and Moons, 2017;Dong et al., 2018;Lichak et al., 2022). Furthermore, the possibility of zoonotic transmission (see Ziarati et al., 2022) makes these infections not only a major challenge for the farming of betta fish but also a public health concern. ...
Siamese fighting fish (Betta splendens) play an important role in the global aquarium trade, but their susceptibility to mycobacteriosis could challenge the long-term sustainability of the industry. Thus, this study aimed to characterize rapidly growing non-tuberculous mycobacteria (RGM) isolated from the fish and to investigate their pathogenicity. Five RGM species were investigated, namely Mycobacterium chelonae, M. cosmeticum, M. farcino-genes, M. mucogenicum, and M. senegalense. The isolates were phenotypically and biochemically characterized and assessed for susceptibility to 18 antibiotics and five disinfectants. The pathogenicity of the isolated bacteria was evaluated through experimental infection of the fish by intraperitoneal injection. The results of phenotypic and biochemical typing allowed the identification of some of the isolates and provided preliminary information to distinguish between the bacteria. All isolates were resistant to at least four antibiotics, with multiple antibiotic resistance indexes ranging from 0.22 to 0.61, with M. chelonae having the highest value. Four disinfectants (i.e., ethanol, formalin, chlorine, and povidone-iodine) showed strong antibacterial activity, whereas potassium permanganate proved less effective. An LD 50 value within 7 days showed that M. chelonae had the highest virulence at 7.12 × 10 5 CFU/fish, followed by M. mucogenicum at 4.25 × 10 6 CFU/fish and a range of 1.01-1.65 × 10 7 CFU/fish for the other isolates. Depending on the dose and the isolate administered, some fish displayed acute disease symptoms, while others developed a chronic condition. The acute disease was characterized by short median survival, severe peritonitis, and tissue necrosis. Fish with a chronic infection survived the 42-day trial but were emaciated and had systemic granulomas within their viscera. The findings of this study have not only improved our understanding of the nature of these RGM species but also promoted the development of control strategies to mitigate the negative impact of mycobacteriosis on the Siamese fighting fish industry.
... Spesies ini merupakan kelompok ikan yang memiliki dimorfisme kelamin yang terlihat dari ciri seksual sekunder pada individu jantan dan betina. Ikan cupang jantan lebih digemari karena keindahan warna, bentuk sirip punggung, sirip anal, dan sirip ekornya (Monvises et al. 2009, Wahyudewantoro 2017, Lichak et al. 2022. Karena itu dalam budidaya ikan cupang dapat dilakukan produksi tunggal kelamin (monoseks) jantan untuk memberikan keuntungan. ...
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Betta splendens males have colours and shapes that are popular in the ornamental fish market compared to females. Because of that, betta cultivation can be done through male production using sex reversal technology in directing the sex development of fish to become male (masculinization). The natural ingredient that has been used to masculinize fish is honey. So the research objective was to examine the use of honey by immersing the embryo to masculinize betta. Analyzed masculinization success through characteristics of honey, percentage of male fish, egg hatching rate, mortality every 15 days, and survival at the end of rearing. The embryos used were 20 hours post-fertilization. The research treatment was immersion of betta embryos in honey solution (mL L-1) 5, 10, 15, 20, and 25. Soaking was carried out for 7 hours. The results showed that the honey used had 0.31% potassium and a pH of 4. In this study, the administration of honey did not affect the number of male betta. Giving honey 25 mL L-1 water produced 56.98 ± 4.58% of males, egg hatching rate 99.17 ± 1.67%, and survival at 90 days after hatching 79.89 ± 4.50%. Mortality occurs at the start of larval rearing. After the age of 60 days after hatching, there is no death in betta. The high values of egg hatching and survival rates indicate that honey is a natural material safe for masculinizing fish in mono-sex aquaculture.
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Siamese fighting (betta) fish are among the most popular and morphologically diverse pet fish, but the genetic bases of their domestication and phenotypic diversification are largely unknown. We assembled de novo the genome of a wild Betta splendens and whole-genome sequenced 98 individuals across five closely related species. We find evidence of bidirectional hybridization between domesticated ornamental betta and other wild Betta species. We discover dmrt1 as the main sex determination gene in ornamental betta and that it has lower penetrance in wild B. splendens . Furthermore, we find genes with signatures of recent, strong selection that have large effects on color in specific parts of the body or on the shape of individual fins and that most are unlinked. Our results demonstrate how simple genetic architectures paired with anatomical modularity can lead to vast phenotypic diversity generated during animal domestication and launch betta as a powerful new system for evolutionary genetics.
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Background Fishes are the one of the most diverse groups of animals with respect to their modes of sex determination, providing unique models for uncovering the evolutionary and molecular mechanisms underlying sex determination and reversal. Here, we have investigated how sex is determined in a species of both commercial and ecological importance, the Siamese fighting fish Betta splendens . Results We conducted association mapping on four commercial and two wild populations of B. splendens . In three of the four commercial populations, the master sex determining (MSD) locus was found to be located in a region of ~ 80 kb on LG2 which harbours five protein coding genes, including dmrt1 , a gene involved in male sex determination in different animal taxa. In these fish, dmrt1 shows a male-biased gonadal expression from undifferentiated stages to adult organs and the knockout of this gene resulted in ovarian development in XY genotypes. Genome sequencing of XX and YY genotypes identified a transposon, drbx1 , inserted into the fourth intron of the X-linked dmrt1 allele. Methylation assays revealed that epigenetic changes induced by drbx1 spread out to the promoter region of dmrt1 . In addition, drbx1 being inserted between two closely linked cis -regulatory elements reduced their enhancer activities. Thus, epigenetic changes, induced by drbx1 , contribute to the reduced expression of the X-linked dmrt1 allele, leading to female development. This represents a previously undescribed solution in animals relying on dmrt1 function for sex determination. Differentiation between the X and Y chromosomes is limited to a small region of ~ 200 kb surrounding the MSD gene. Recombination suppression spread slightly out of the SD locus. However, this mechanism was not found in the fourth commercial stock we studied, or in the two wild populations analysed, suggesting that it originated recently during domestication. Conclusions Taken together, our data provide novel insights into the role of epigenetic regulation of dmrt1 in sex determination and turnover of SD systems and suggest that fighting fish are a suitable model to study the initial stages of sex chromosome evolution.
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The present study evaluated the influence of thermal management during the larviculture of Betta splendens on survival and sex ratio, aiming to increase the proportion of males. Newly hatched larvae were subjected to different thermal regimes, namely, T25, T28, T30 and T33 (25, 28, 30 and 33ºC, respectively). The experiment was laid out in a completely randomized design, with 4 treatments and 10 repetitions. Thermal treatment was maintained until 15 days post-hatch (DPH). Mortality was determined at the end of the thermal regime and again at 45 DPH. At the end of the experiment, the number of males and females obtained in the different thermal treatments was counted to analyze the obtained sex ratio. There was a significant effect on mortality as a function of temperature only at 15 DPH (p <0.001), with the lowest values recorded in treatments T25, T28 and T30. In terms of sex ratio, up to 65% of males were obtained in treatment T33 (p = 0.037). In conclusion, thermal management during the larval period can be a strategy to increase the proportion of males, but the increase in mortality due to the rise in temperature should be considered.
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Resolving the genomic basis underlying phenotypic variations is a question of great importance in evolutionary biology. However, understanding how genotypes determine the phenotypes is still challenging. Centuries of artificial selective breeding for beauty and aggression resulted in a plethora of colors, long fin varieties, and hyper-aggressive behavior in the air-breathing Siamese fighting fish (Betta splendens), supplying an excellent system for studying the genomic basis of phenotypic variations. Combining whole genome sequencing, QTL mapping, genome-wide association studies and genome editing, we investigated the genomic basis of huge morphological variation in fins and striking differences in coloration in the fighting fish. Results revealed that the double tail, elephant ear, albino and fin spot mutants each were determined by single major-effect loci. The elephant ear phenotype was likely related to differential expression of a potassium ion channel gene, kcnh8. The albinotic phenotype was likely linked to a cis-regulatory element acting on the mitfa gene and the double tail mutant was suggested to be caused by a deletion in a zic1/zic4 co-enhancer. Our data highlight that major loci and cis-regulatory elements play important roles in bringing about phenotypic innovations and establish Bettas as new powerful model to study the genomic basis of evolved changes.
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The complete mitochondrial genome (mitogenome) of the peaceful betta (Betta imbellis) was obtained using next-generation sequencing. The sample of B. imbellis was collected from its native habitat in Southern Thailand. The mitogenome sequence was 16,897 bp in length, containing 37 genes with identical order to most teleost mitogenomes. Overall nucleotide base composition of the complete mitogenome was determined as AT bias. Phylogenetic analysis of B. imbellis showed a closer relationship with bubble-nesting fighting fish. This annotated mitogenome reference can be utilized as a bioresource for phylogenetic studies to support betta conservation programs.
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Mahachai bettas (Betta mahachaiensis) are distributed in areas of brackish water with Nipa Palms in Samut Sakhon, Thailand but urbanization is restricting their biodiversity. A complete mitochondrial genome (mitogenome) of B. mahachaiensis was determined to support conservation programs. Mitogenome sequences were 16,980 bp in length with slight AT bias (61.91%), containing 37 genes with identical order to most teleost mitogenomes. Phylogenetic analysis of B. mahachaiensis showed a closer relationship with B. splendens. Results will allow the creation of a reference annotated genome that can be utilized to sustain biodiversity and eco-management of the betta to improve conservation programs.
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Conspecific male animals fight for resources such as food and mating opportunities but typically stop fighting after assessing their relative fighting abilities to avoid serious injuries. Physiologically, how the fighting behavior is controlled remains unknown. Using the fighting fish Betta splendens, we studied behavioral and brain-transcriptomic changes during the fight between the two opponents. At the behavioral level, surface-breathing, and biting/striking occurred only during intervals between mouth-locking. Eventually, the behaviors of the two opponents became synchronized, with each pair showing a unique behavioral pattern. At the physiological level, we examined the expression patterns of 23,306 brain transcripts using RNA-sequencing data from brains of fighting pairs after a 20-min (D20) and a 60-min (D60) fight. The two opponents in each D60 fighting pair showed a strong gene expression correlation, whereas those in D20 fighting pairs showed a weak correlation. Moreover, each fighting pair in the D60 group showed pair-specific gene expression patterns in a grade of membership analysis (GoM) and were grouped as a pair in the heatmap clustering. The observed pair-specific individualization in brain-transcriptomic synchronization (PIBS) suggested that this synchronization provides a physiological basis for the behavioral synchronization. An analysis using the synchronized genes in fighting pairs of the D60 group found genes enriched for ion transport, synaptic function, and learning and memory. Brain-transcriptomic synchronization could be a general phenomenon and may provide a new cornerstone with which to investigate coordinating and sustaining social interactions between two interacting partners of vertebrates.
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Ever decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish ( Betta splendens ) that was carried out during a practical university master's course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behaviour. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published Hi-C data. The use of ~35x nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using the Hi-C data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 95.8% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly. We present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university master's course. The use of ~35× long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.
The role of hormones as modulators of aggressive behavior in fish remains poorly understood. Androgens and corticosteroids, in particular, have been associated with aggressive behavior in fish but it is still not clear if animals adjust the secretion of these hormones to regulate behavior during ongoing fights, in response to fight outcomes in order to adjust aggressive behavior in subsequent fights, or both. With its stereotyped displays and high aggression levels, the Siamese fighting fish Betta splendens is an excellent model to investigate this question. Here, we compared the behavioral and endocrine response of male B. splendens to fights where there is no winner or loser by presenting them with a size-matched live interacting conspecific behind a transparent partition or with a mirror image. The aggressive response started with threat displays that were overall similar in frequency and duration towards both types of stimuli. Fights transitioned to overt attacks and interacting with a live conspecific elicited a higher frequency of attempted bites and head hits, as compared with the mirror image. There was a pronounced increase in plasma androgens (11-ketotestosterone and testosterone) and corticosteroids (cortisol) levels in response to the aggression challenge, independent of stimulus type. Post-fight intra-group levels of these hormones did not correlate with measures of physical activity or aggressive behavior. A linear discriminant analysis including all behavioral and endocrine data was a poor classifier of fish from the conspecific and mirror trials, showing that overall the behavioral and endocrine response to mirror images and conspecifics was similar. The results show that fight resolution is not necessary to induce an evident increase in peripheral levels of androgens and corticosteroids in B. splendens. However, the function of these hormones during present and future aggressive contests remains to be clarified.