Introduction:
Regeneration
Regeneration is a crucial part of life for all organisms. It ranges from cell renewal
to organ regeneration using a range of processes of regrowth (Kawakami, 2010). In
mammals, cells must regenerate to close wounds or repair organs, but some other species
have harnessed regeneration so well that they can replace severed limbs and/or body
parts. While these capabilities vary greatly between different species, several are widely
known for their advanced regenerative capabilities. These include hydra, axolotl,
freshwater planarians, and zebrafish. Because of their advanced capabilities, these species
are probably the most widely used model organisms to study regeneration (National
Institute, 2020; Tsai, 2020).
Planarian Regeneration
Planarians are members of a phylum of freshwater flatworms called the
Platyhelminthes. This family of worms is not parasitic, but they do possess complex
nervous, intestinal, musculature, and reproductive systems. In addition, they are
symmetrical organisms that feed using the complex structure of a pharynx and use
primitively developed eyes on the dorsal surface of their head to sense light in their
environment (Ivankovic, et al., 2019). Despite all these advanced features in such
primitive looking organisms, some species of planarians are known to have impressive
regenerative abilities. Planarians only require 1/129th of their original number of cells, or
about 10,000 cells, to regenerate the rest of their body (Lobo, et al., 2012; Ivankovic, et
al., 2019). In fact, planarians reproduce asexually by basically ripping themselves in half
and regenerating the opposing half of their body with their own cells. Planarians consist
of 20-30% neoblast cells, which are stem cells specific to these species. These neoblasts
are key to their regenerative abilities and can differentiate into any kind of adult cell via
the blastema that forms over a wound. What is uncertain is how those cells are signaled
to migrate and differentiate into complex structures with specific functions (Ivankovic, et
al., 2019).
The exact processes used in tissue regeneration has eluded scientists for years, but
significant progress has been made. Once a wound is formed, planarians can harness the
genes they used during embryonic development to begin expression again and reform the
part of their body that has been injured (Pfefferli & Jaźwińska, 2015). After a small
injury, a cascade of signals activates the localized cells to close and slightly regrow the
wound. After a large injury, which resulted in a loss of the planarian’s tissue, a global
cascade of signals is initiated to begin wound closure and regeneration. Signaling of
extracellular signal-related kinase (ERK) lead to blastema formation within only 30-45
minutes after the injury. Once the wound is closed and protected from the outside world,
apoptosis is initiated at wound site to rid the body of cells that are too far damaged for
repair. This lasts up to 4 hours after the initial injury, followed by neoblast mitosis
throughout the planarian’s body. Next, the neoblasts migrate to the wound site and begin
mitosis again, but this time the mitosis would occur only locally for up to 72 hours after

the initial injury. Once again apoptosis occurs, but this time it is globally. Finally, the
tissue orientation, regrowth, and patterning can begin and last for up to two weeks after
the initial injury.
The regeneration orientation process is communicated throughout the body using
morphogen and chemical gradients and pathways. Morphogens are proteins generally
responsible for cell differentiation. The concentration of morphogen that a cell receives is
the determining factor for what cell it should
differentiate into. A global morphogen gradient
is expressed throughout the entire organism,
while a local morphogen gradient is expressed
only in a certain location or type of cells, not the
entire organism. Differences in morphogen
gradient expression are regulated by
developmental regulatory genes. Sometimes
regeneration is due to one morphogen with a
globalized gradient, other times it is due to
Figure 1. Example of a general
morphogen gradient. The different colors
multiple localized signals from different
of cells on the bottom represent the
chemical pathways. Moreover, gap junctions can
different concentration thresholds that
allow cell-to-cell signaling after being
will each produce a different cell type
stimulated by the body’s bioelectric or nervous
(Alnaif & Lander, 2017).
system. Gradients have been found to be crucial
in the orientation of the regenerating tissue.
Anterior/posterior polarity is established using several different gradients at the
opposing ends of the planarian. The posterior is established by Hedgehog, Wnt, and bcatenini pathways. Several studies have shown that if any of these pathways become
inhibited, a head will develop in the posterior axis rather than a tail (Lobo, et al., 2012;
Gurley, et al., 2008; Peterson & Reddien, 2011). Since head polarity is the global default,
the Wnt and b-catenini inhibitor notum is needed at the head to block the Wnt and betacatenin gradient signals to produce a head at the anterior
axis. Exogenous application of retinoic acid has also
been found to be a crucial part of establishing the
anterior axis, and without either of these signals, a tail
will develop anteriorly (Lobo et al., 2012; Iglesias, et al.,
2011; Romero & Bueno, 2001). Another essential
Figure 2. Axes of a planaria. (A)
element for head formation is bioelectric stimulation.
Anterior end. (P) Posterior end. (D)
Scientists are unsure why, but several studies show that
Dorsal side. (V) Ventral side. (M)
without this stimulation at the anterior axis, a tail will
Medial, middle body. (L) Lateral body.
form, despite the signaling pathways still being complete (Lobo, et al., 2012)
(Dimmitt & Marsh, 1952; Lobo, et al., 2012; Marsh &
Beams, 1952).

Similarly, dorsal/ventral polarity is also established due to several global protein
gradients. The first is a bone morphogenetic protein (BMP) gradient. In vertebrates the
origin of this gradient establishes the ventral axis, whereas in invertebrates, like
planarians, the BMP gradient origin establishes the dorsal axis (Brown, et al., 2008;
Lobo, et al., 2012; Lowe, et al., 2006). Anti-dorsalizing morphogenetic protein (ADMP)
produces a gradient opposite of the BMP gradient to establish the proper opposing end.
For vertebrates this would define the dorsal axis, and for invertebrates, like planarians,
this would define the ventral axis. The ADMP gradient is the default dorso-ventral axis
gradient within developing and regenerating organisms since it is inhibited by excess
BMP molecules, and without them, neither end of the axis would be different from the
other. For instance, without BMP inhibition in invertebrates, two ventral axes would form
by default rather than one dorsal axis and one ventral axis. Moreover, the BMP inhibitor
protein noggin is needed at the same end as the ADMP gradient to inhibit the BMP
gradient and establish the proper end. Silencing any of these three genes disrupts dorsoventral polarity throughout the organism (Gavino & Reddien, 2011; Lobo, et al., 2012;
Molina, et al., 2011). Thus, the three molecules essentially work in a negative feedback
loop to establish dorso-ventral axis polarity.
Medial/lateral axis polarity is established through protein gradient in regenerating
tissue. The slit gene family is expressed along the medial line and the Wnt5 gene family
is expressed along lateral line to keep the slit gradient at bay (Adell, et al., 2009; Lobo et
al., 2012; Gurley, et al., 2010). Inhibition of either of these gradients results in axis
collapse as well as nervous system collapse. The medial/lateral protein gradient has been
found to work in close association with the dorso-ventral gradient. Scientists are unsure
why but disrupting the ADMP gradient also inhibits the Wnt5 expression gradient,
disabling the lateral axis and producing multiple pharynxes in the planarian (Adell, et al.,
2009; Lobo et al., 2012; Gavino & Reddien, 2011; Gurley, et al., 2010).
Following tissue orientation, tissue differentiation is initiated and orchestrated by
cellular communications between the blastema, old tissues, and new tissues (Lobo, et al.,
2012). Less is known about how tissue identity is determined than the tissue orientation
pathways. One family of genes called piwi are thought to maintain of neoblast stem cells
and prevent their differentiation throughout the body so that it is ready to repair itself
whenever an injury occurs. Several different molecular markers have been identified in
neoblasts before and after they have differentiated, suggesting that neoblasts are
somewhat pre-determined before regeneration is initiated. Inhibition of this pathway
results in a loss of regenerative abilities (Eisenhoffer, et al., 2008; Lobo et al., 2012;
Oviedo & Levin, 2007; Palakodeti, et al., 2008; Reddien, et al., 2005). Additionally, the
homologous proteins phosphatase and tension (PTEN) are present in planarians to
regulate neoblast activity and prevent the stem cells from hyperproliferating. The proteins
essentially act as tumor suppressors, as inactivation of either results in abnormal growths
throughout the organism’s body (Lobo et al., 2012; Oviedo & Levin, 2007). Otherwise,
there is little sound evidence of how tissue identity is established in planarian
regeneration.

Zebrafish Regeneration
In addition to planarian, the zebrafish, Danio rerio, is a common model organism
for studying regeneration. Zebrafish are known to regenerate essential tissues in their
bodies including their fins, heart muscle, and nervous system cells (Pfefferli &
Jaźwińska, 2015; Qin, et al., 2009). Besides zebrafish being a great vertebrate model
organism, they also share seventy percent of their genome with humans, meaning that
many of the discoveries made about their genomic regulation is relevant to and has the
possibility to be harnessed in humans as well (Gilbert, 2016). Unlike humans, zebrafish
grow throughout adulthood, so their caudal fins would continue growth whether cut or
not. Additionally, their bones grow and regenerate from the distal tip, not the proximal
width like in humans (Pfefferli & Jaźwińska, 2015). In other words, their bones grow by
adding new tissue to the farthest tip of the existing bone, whereas human bone tissue is
produced and added to closest tip of the existing bone. The most studied part of the
zebrafish is by far its caudal fin regeneration, in part because it regrows faster than any
other fin out of necessity to the organism’s survival (Pfefferli & Jaźwińska, 2015). This
regeneration includes the orchestration of at least 3 different tissues, including bone.
What sets zebrafish apart from other model organisms is the symmetrical morphology of
their tail as well as its accessibility for amputation, tests, and photography. Like planaria,
zebrafish regeneration is classified as epimorphic, meaning proliferation of tissue
materials occurs before the new body part begins development (Kawakami, 2010;
Pfefferli & Jaźwińska, 2015; Tsai, 2020).
A similar stepwise process of regeneration is used in zebrafish as in planaria.
Directly after a tissue loss injury, fibroblasts, highly proliferative undifferentiated
mesenchyme cells, begin proliferation and migration to the wound site. Dedifferentiated
osteoblasts follow, and both cell types begin blastema formation. Within a day the
blastema is formed to protect the wound from the external environment as well as cells
organize regeneration between new and old tissues. Once the blastema is formed,
fibroblast cells produce a protein called tenascin C to help organize and thicken its tissues
(Jaźwińska, et al., 2007; Pfefferli & Jaźwińska, 2015). Directly under the blastema, a
wound epithelium begins to form. The two layers are thought to work in conjunction to
regenerate the severed tissues.
The wound epithelium and blastema work closely to organize mesenchymal and
osteoblast cell proliferation and differentiation. The blastema has been shown to secrete
proteins such as Fgf20a, Sdf1, Igf2b, and retinoic acid to organize the formation of the
wound epithelium (Blum & Begemann, 2013; Bouzaffour et al., 2009; Chablais &
Jaźwińska, 2010; Dufourcq & Vriz, 2006; Pfefferli & Jaźwińska, 2015; White et al.,
2005; Whitehead et al., 1994). Once formed, the wound epithelium has been found to
secrete proteins such as Sonic hedgehog (Shh), Wnt5b, and Fgf24 to help orient the
blastema for proliferation (Laforest et al., 1998; Lee et al., 2009; Pfefferli & Jaźwińska,
2015; Poss et al., 2000; Quint et al., 2002). The feedback cues are thought to help create
gradients for the proliferating cells to regenerate in the correct orientation along each

axis. This process is nearly the same pathway that is exhibited in planaria, showing the
conservation of processes between species in nature.

Figure 3. Pink staining shows the original tail while the blue staining shows the bone regrowth. (A)
Regenerating zebrafish fin. (B) Close up of the transition area from original fin to new growth. (C)
Brush-like spindles, actinotrichia, extending from the blue stained regrowth. (D) Thickened proximal
bone of tail versus the thinner distal bone shown in (B) (Pfefferli & Jaźwińska, 2015).

The next two days are then defined by blastema outgrowth as fin skeleton
formation begins. The fin skeleton can be seen in Figure 3 below. First, actinotrichia,
non-mineralized spicule segments, are extended from the original bone tissue. These
segments are organized in brush-like bundles at the tip of the old bone to act as
architecture for the new osteoblasts (Duran´ et al. 2011; Kawakami, 2010; Knopf et al.
2011; Pfefferli & Jaźwińska, 2015). The protein secretions Shh and BMP from the wound
epithelium have been found to be instrumental in guiding pre-osteoblasts to reform the
pattern of mature bones (Laforest et al., 1998; Pfefferli & Jaźwińska, 2015; Smith et al.,
2006; Quint et al., 2002; Zhang et al., 2012). One study has found that several
actinotrichia genes are activated to direct the tissue extension, including the gene
actinodin1. However, unlike in embryonic development or human bone, the growth is
extended distally, rather than proximally. In addition, the surrounding tissues are also
organized and extended as the fin becomes vascularized and innervated the farther the
blastema extends (Bayliss et al. 2006; Pfefferli & Jaźwińska, 2015).
Following bone regeneration, the apical blastema begins extension. Notch
signaling organizes undifferentiated mesenchymal cells during the blastema outgrowth
(Grotek et al., 2013; Munch et al., 2013; Pfefferli & Jaźwińska, 2015). The blastema
itself is thought to be an upstream organizer for the tissues, influencing cell proliferation,
epidermal patterning, and cell redifferentiation with Wnt signaling. Moreover, Fgf and
BMP signals from the blastema are believed to be responsible for coordinating secondary
osteoblast maturation (Pfefferli & Jaźwińska, 2015; Wehner et al. 2014).

Uncut

1 dpa

3 dpa

6 dpa

12 dpa

20 dpa

Figure 4. Progression of tail regeneration in a zebrafish in days post amputation (dpa). 1dpa shows the
formation of the wound epidermis. The thick white end of the tail shown 3dpa is the fully formed blastema.
The blastema outgrowth is shown at 6pda and 12dpa as more advanced. 20dpa shows the fully regenerated
zebrafish tail (Pfefferli & Jaźwińska, 2015).

The next three days, the blastema begins to shrink and ends up covering only the distal
most tip of the tail. This whitish tip will remain on the zebrafish’s’ tail for the rest of its
life as a scar would on human skin. The proximal most undifferentiated cells begin
differentiation into their destined cell fates. Several studies have shown that, like
planarian neoblasts, undifferentiated cells have morphogenetic markers in their DNA that
can be traced to their future, differentiated, cell type. This growth and repatterning of new
and old tissue continues for up to two to three weeks until the zebrafish fin is fully
regenerated and functional (Knopf et al. 2011; Pfefferli & Jaźwińska, 2015; Singh et al.
2012; Sousa et al. 2011; Stewart & Stankunas 2012; Tu & Johnson 2011).
Epigenetic Influence
For years, the mechanisms behind the variation in regenerative abilities between
species has eluded the scientific community. However, recent findings show that the
variation in regenerative abilities may be linked to different epigenetic modifications
found in each species’
genome. Epigenetic
modifications are
defined as “a stably
heritable phenotype
resulting from changes
in a chromosome

Figure 5. Chromatin structures where epigenetic modifications are
applied (What is epigenetics, n.d.).

without alterations in the DNA sequence” (Berger et
al., 2009). These changes in the chromosome can come
in several different forms including DNA acetylation
and methylation. DNA acetylation is considered the
default state of chromatin where acetyl groups are
attached to the histone proteins chromatin is wrapped

Figure 6. Visual difference between
euchromatin and heterochromatin (Cornell,
2016).

around (Sharon, 2017). This type of chromatin is loose, easily transcribable, and called
euchromatin (Javaid & Choi, 2017). Conversely, DNA methylation is defined as the
attachment of a methyl group to a histone protein or a nucleotide base in the DNA
sequence (Mulligan, 2016; Sharon, 2017). The addition of this methyl group changes
how the DNA is packaged. Hypermethylation causes tightly packed, harder to transcribe
chromatin called heterochromatin (Javaid & Choi, 2017). Both can result in a difference
in how the DNA is transcribed and can be detrimental to an organisms’ health depending
on the gene(s) that are methylated (Dincer, 2016).
Based on one study of gene 5-methyl-cytosine and 5-hydromethylcytosine
comparisons, early phase regrowth in regenerative species is characterized by DNA
demethylation and expression of repair-related genes (Hirose et al. 2013; Pfefferli &
Jaźwińska, 2015). Additionally, it theorized that these genes are upregulated in the
presence of an injury, and downregulated once regeneration has ceased (Rodriguez &
Kang, 2020). One study provides evidence that histone modifications at specific loci, like
the demethylation of the H3K27me3 gene, can re-activate the genes necessary to
regenerate living structure like all organisms once did in embryonic development
(Pfefferli et al., 2014; Pfefferli & Jaźwińska, 2015; Stewart, et al., 2012). Further, several
genes related to the nucleosome remodeling and deacetylase (NuRD) complex were
found to be upregulated during blastema proliferation. Several of the genes required for
upregulation to complete this complex are the chd4a, hdac1, rbb4, and mta2 genes.
Failure of this complex to activate results in no formation of the actinotrichia bristles, and
thus, no architecture for the undifferentiated cells to regenerate upon (Pfefferli et al.,
2014; Pfefferli & Jaźwińska, 2015). Hence, epigenetic variation plays a profound role in
variation of regenerative abilities between organisms.
Regenerative Abilities of Fish
Overall, the regeneration process differs greatly between different types of
organisms. Nevertheless, many kinds of fish have been used as model organisms for
studying regeneration since the 1700s. The regeneration process between many kinds of
fish is remarkably similar (Kawakami, 2010). As most research on fish fin regeneration
has been focused on defining the specific steps of the regeneration process, how well the
regrowth matches the original fin in shape, size, and coloration has often not been
examined. Most studies that have been conducted utilize fish with simple fin shape and
coloration, such as zebrafish. The aim of this experiment was to examine fin regeneration
in more elaborate fish fins to see how the regrowth compared to fish with a simpler fin
shape and coloration. Thus, the subject of study chosen for this experiment was the
species of Betta splendens. This species is known for its elaborate tail shapes and
coloration, but not necessarily its research value. While there is little to no research on
regeneration in this species, these fish do possess the regenerative properties necessary to
regrow large parts of their fins after an injury. However, they are often used as a model
organism to study behavior and provide an opportunity to also examine whether fin
regeneration affects behavioral displays and their epigenetics.

Aggressive Behavior of Male Betta splendens
Male Betta splendens, also known as Siamese fighting fish, are widely known for
their displays of aggressive behavior. Whether exhibiting courtship behavior or general
aggressive behavior towards another male, Betta splendens have been used for scientific
behavioral studies for a variety of reasons. For instance, despite having to be kept in
separate tanks, they are relatively easy to care for, and their behavioral aggressions are
easy to count and identify in behavioral test (Todd, et al., 2008).
In the wild, a Betta splendens’ fitness relies heavily upon dominance over other
males in its species. Often, wild male Betta splendens form hierarchies as ranking of the
fittest individual to the least fit individual (Jameson, et al., 1999). Fitness hierarchies are
developed based on female sexual selection as well as aggressive competition between
males (Jameson, et al., 1999;
Milinski, 2014). Further, aggressive
behavior is also used to defend
territory and nests to help ensure
their offspring survive (Forsatkar, et
al., 2017; Todd, et al., 2008). Male
Betta splendens aggressive
behaviors are used scare off and
Frontside
Broadside
fight their opponents since the
Figure 7. Visual orientations of Betta splendens
behaviors generally make them look
larger and fiercer (Todd, et al., 2008; Qvarnström and Forsgren, 1998). Betta splendens
aggressive behaviors generally consist of frontside or broadside movements. Frontside
movements include operculum and branchiostegal membrane flaring, biting, and pectoral
fin beating (Glesener, 2001; Simpson, 1968; Todd, et al., 2008). Broadside movements
include tail beating, pelvic fin flickering, and tail flashing (McGregor, et al., 2001;
Simpson, 1968; Todd, et al., 2008). Other movements that are also considered aggressive,
but are not categorized as either frontal or broadside, include chasing and charging the
opponent (Halperin, et al., 1997; Simpson, 1968; Todd, et al., 2008). Though not as
common, similar aggressions have been examined in female Betta splendens (Simpson,
1968; Todd, et al., 2008). Thus, it makes sense that both sexes display the same behaviors
in captivity because they have evolved so that their reproductive success depends on it.
Numerous studies have shown that male Betta splendens aggressive behaviors are
affected by the presences of a female (Forsatkar, et al., 2017; Lück, 2014; Milinski, 2014;
Todd, et al., 2008). Further, there is some research available presenting numerous effects
of stress on fish behavior. For example, salmonoids generally present proactive,
aggressive behaviors or reactive, shyer behaviors when they are stressed (Laursen et al.,
2011). In zebrafish, stressed behavior is presented as swimming or sitting on the bottom
of the tank (Valvarce, et al., 2020). However, there is little to no research looking at how
aggressive behaviors in male Betta splendens are affected by stress and whether
regenerated fins are functionally normal in behavioral displays. As part of this

experiment, behavioral tests were conducted with male bettas that did and did not have
their tails cut for regenerative study.
Experimental Aims
This study aims to determine whether fin regeneration affects male Betta
splendens aggressive behavioral displays. Tail amputation is thought to affect either the
number of times or the amount of time aggressive behaviors are displayed because
aggressive behavioral displays are heavily reliant on the use of their tail. Since external
environment can also shape epigenetics, this study also aims to examine whether tail
regeneration results in epigenetic changes to DNA methylation.
Materials/Methods:
Experimental Setup & Maintenance
Unforeseen circumstances forced this experiment to be conducted from a home
setting rather than in a laboratory. Betta splendens and the materials for maintenance,
care, and experimentation were dropped off at my house by my professor periodically
when it was time for me to move to the next step in my experiment.
Ten, red, male Betta splendens were bought from a local pet store and delivered to
my house over the course of three weeks. Each was labeled with a number 1-10 to for
organizational data purposes. To set up their tanks, one gallon of tap water was treated
with 5mL of Aqueon, Betta Bowl Plus to dechlorinate the water. It was left to sit open in
an empty bedroom for at least 24 hours to remove any excess minerals in the water, and
to allow it to reach room temperature. Then, in a bathroom, an empty one-gallon tank was
filled with the dechlorinated water and a Betta splenden, still in its small transportation
bowl, was placed in the tank to acclimate. After at least an hour, the fish was gently
submerged into the tank and the small transportation bowl was removed. This process
was repeated nine times as new fish were acquired.
Eight of the nine tanks used were small, clear, rectangular tanks with an
individual fish in them. The nineth tank was slightly larger and housed two Betta
splendens, one of which had its tail cut, and the other of which did not. Five of the nine
tanks were then placed on its own shelf on a shelving unit in the corner of my bedroom
for storage, to prevent the tanks from shaking, and to prevent the fish from seeing each
other. The other four tanks were placed by twos on another, smaller shelving unit in a
corner right outside of my bedroom. Pieces of cardboard were placed in between the two
tanks on each shelf to prevent the fish from constantly seeing each other and displaying
aggressive behaviors towards its neighbor.
The Betta splendens were fed 2-3 TopFin color enhancing betta bits at
approximately 8AM and 8PM, five days a week. On the weekends, they were not fed at
all as to help prevent overfeeding. Once a week, debris and 20-30% of every tank’s water
was removed and replaced with the same dechlorinated tap water as the tank was started
with.

Anesthetization & Amputation
After three weeks of adapting to the tanks and house, 5 of the 10 Betta splendens
had their tails cut. The 5 fish amputated were randomly picked from the 10 males in the
experiment. While their tail lengths were not precisely measured, all males and their tales
were relatively the same size. To amputate, my professor dropped off Tricaine-S (MS222), 1 pair of forceps, sterile razor blades, a petri dish, a box of slides, and a box of slide
covers. A folding table in an empty living room of the house next to a window so that
there was sufficient light. It was covered with a sheet of industrial plastic for sterility and
easy clean-up. One fish, in its tank, was set on the card table as well as the rest of the
supplies. The MS-222 was poured into one of the small transportation bowls, and the fish
was caught in its tank using another. The fish was then gently transferred to the bowl of
MS-222 and allowed in the solution until just limp and anesthetized. This process took
significantly longer, up to 2-3 minutes, than for the zebrafish model organisms because of
how much larger the Betta splendens are in size. A new, sterile, plastic spoon was used to
scoop the limp fish out of the anesthetic and gently place on the petri dish. Then, the tail
was spread using forceps, and a cut of approximately one third of the tail was made using
a new, sterile razor blade. Using the same spoon, the fish was then gently placed back
into its tank and water wash pushed over its gills to help it recover. Once the fish began
showing signs of movement again, forceps were used to transfer the sample from the
petri dish to the microscope slide. Once again, forceps were used to position the sample
and place the cover slip over it. This process was repeated for each of the 5 Betta
splendens with the same equipment.
Tail Amputation & Behavioral Test Schedules
Since the fish were received in three staggered groups, their
tails were cut at staggered timeframes. This meant that the final tail
cuts of all 5 fish showed 3 different weeks of the regrowth process.
The final tail cuts were made 6 weeks after the first fish’s tail was
cut, meaning the samples show tail regrowth of 4, 5, and 6 weeks.
There were two additional samples taken from fish whose tails were
not previously cut to use as a control. The tail cut schedule can be
seen in Figure 8.

Cut Tails
R2’
R3
R5
R8
R9
Uncut Tails
R1
R4
R6
R7
R10

Tail Cut Schedule
Date First Cut: Date Second Cut:
9/25/2020
9/18/2020
9/25/2020
10/2/2020
10/2/2020

10/30/2020
10/30/2020
10/30/2020
10/30/2020
10/30/2020

~
~
10/30/2020
~
10/30/2020

~
~
~
~
~

Figure 8. Tail cut schedule.

Behavioral tests were conducted 3 weeks after the tails were cut. Even at this
stage, the tail regrowth in the bettas was not fully complete
Behavioral Test Schedule
but needed to be conducted for the rest of the data to be
Dates:
Uncut: Uncut
10/9/2020
R1:R4
collected within the semester. Figure 9 shows the schedule of
10/16/2020 R1:R6
when each fish was tested against each group. Each group of
10/23/2020 R7:R10
fish was tested against another of its group and another of the
Uncut: Cut
opposite group. For each test, the fish were placed next to
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R4:R3
each other in their separate, clear tanks for 5 minutes. The
10/16/2020 R1:R5
tests were recorded and later analyzed for the number of times
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and the amount of time, in seconds, each aggressive behavior
10/23/2020 R7:R8
was displayed. Tail beating, tail flashing, flaring gills,
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extending the gill membrane, raising the dorsal fin, lowering
Cut: Cut
the head, darting toward the opponent, and nipping at the
10/9/2020
R3:R2’
opponent were all considered aggressive displays. Nipping at
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the opponent was only examined for the number of times it
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occurred and not the amount of time it occurred because
Figure 9. Behavioral test
nipping happens so quickly it is impossible to count the length
schedule. Red indicates
which fish were analyzed in
of time that it occurred. The number of aggressive displays
each test.
between the beginning and ending each test were not
accounted for.
DNA Isolation
The second set of tail samples taken from the group of males with cut tails as well
as the only tail samples taken from the males with previously uncut tails were frozen to
40°C within an hour after amputation. The samples were chopped into the smallest pieces
possible on the slides they were stored on using a sterile razor blade for each sample to
not contaminate any samples. DNA was extracted using the DNEasy DNA extraction kit
(Quiagen). The procedure was modified to extend the 56°C incubation to two hours, and
the final eluded in 100μl rather than the time and amount described in the kit. This was to
ensure the thicker sample fully broke down and to increase the DNA concentration.
DNA Purification & Analysis
The original samples of extracted DNA were prepared and tested for the amount
of DNA in each sample using spectrophotometer. However, only incorrect readings from
the machine were acquired because all the transmittance levels were near 3, which is too
high of a reading for just a blank sample containing deionized water, let alone samples
containing DNA. An ethanol precipitation with Sodium Acetate was performed on the
samples with DNA, to further purify the DNA, in case contamination was an issue. While
this slightly decreases the amount of DNA that will be in each sample, it ensure the DNA
is much purer. After re-testing the machine, the same results, that were too high, were
given again. Originally an epigenetic analysis of the amount of methylation in each
sample was supposed to be conducted. However, since the spectrophotometer
malfunctioned, so the amount of DNA in each sample was not able to be obtained,

meaning the epigenetic tests could not be run to find the percentage of methylation in
each.
Statistical Analyses
The data collected from the behavioral tests was first analyzed using a ShapiroWilk Test of Normality. Four of the sixteen groups of the number of times aggressive
behavior was displayed were found to be statistically significant from normal as shown in
Table 1. The rest of the data sets, in both the number of times and the amount of time
groups, were found to be of normal distribution and can be found in Tables 1 and 2. To
perform a uniform test on all the data sets in both groups, a nonparametric, two-tailed,
Independent Samples Mann-Whitney-U Test was conducted.
Epigenetic tests were originally supposed to be conducted on the average amount
of methylation in males with both cut and uncut tails. Those groups were planned to be
tested with the Fisher’s exact test to see if there was a correlation between tail cuts and
amount of epigenetic methylation.
Results:
Aggressive behaviors were examined in a control group of male Betta splendens
with uncut tails, and in an experimental group of males three weeks after caudal fin
amputation. Behaviors were quantified in individual fish, in separate displays when
compared to other control males and when compared to other experimental males. Table
3 below shows the average number of times fish displayed tail beating, tail flashing,
flaring gills, extending the gill membrane, raising the dorsal fin, lowering the head,
darting toward the opponent, and nipping at the opponent. Table 4 below show the
average time fish displayed tail beating, tail flashing, flaring gills, extending the gill
membrane, raising the dorsal fin, lowering the head, and darting toward the opponent.
Normality tests were conducted on each data set within each group to determine the type
of statistical test that should be used to compare data sets. Since several of the data sets
were statistically significant from normal, with a p-value < 0.05, and several others were
close to being statistically significant from normal, with p-values of 0.08, a
nonparametric Mann-Whitney U test was used to compare them. Of the fifteen
comparisons conducted, only two showed a statistically significant difference. The
amount of time the dorsal fin was raised in males with cut tails was significantly higher
than the amount of time the dorsal fin was raised in males with uncut tails, as seen by a pvalue of 0.008 and the bar graph in Figure 19. Similarly, the amount of time males with
cut tails spent lowering their head was significantly greater than the amount of time males
with uncut tails spent lowering their head, shown by a p-value of 0.03 and Figure 21. The
rest of the comparisons showed no statistically significant difference in the number of
times or the amount of time aggressive behavior was displayed between males with cut
and uncut tails, as can be seen in Figures 10-18, 20, and 22-24.

Normality Test Results
Aggressive Display: Tail: Test Statistic:
Tail Beating

Degrees of
P-value
Freedom

Uncut

0.96

5

Cut

0.89

5

0.37

Tail Flashing

Uncut

0.95

5

0.75

Cut

0.96

5

0.80

Flaring Gills

Uncut

0.93

5

0.57

Cut

0.77

5

0.04

Uncut

0.93

5

0.60

Extending Gill
Membrane

0.78

Cut

0.75

5

0.03

Raising Dorsal Fin

Uncut
Cut

0.84
0.88

5
5

0.18
0.30

Lowering Head

Uncut

0.93

5

0.61

Cut

0.92

5

0.56

Uncut

0.80

5

0.08

Cut
Nipping at Opponent Uncut
Cut

0.85
0.95
0.80

5
5
5

0.20

Darting Toward
Opponent

Table 1. Normality Test Results of the number of
times each aggressive behavior was displayed in
males with uncut and cut tails. Yellow highlighting
indicates a p-value calculated from data that is
significantly different from a normal distribution.
Orange highlighting indicates a p-value calculated
from data that is almost significantly different from
a normal distribution.

0.71
0.08

Table 2. Normality Test Results of the amount of
time each aggressive behavior was displayed in
males with uncut and cut tails. None of the data
sets showed a distribution that was statistically
significant from normal.

Normality Test Results
Aggressive Display: Tail: Test Statistic:
Tail Beating
Tail Flashing
Flaring Gills

Extending Gill
Membrane
Raising Dorsal Fin
Lowering Head
Darting Toward
Opponent

Aggressive Display:

Degrees of
P-value
Freedom

Uncut
Cut
Uncut
Cut
Uncut

0.84
0.92
0.89
0.92
0.93

5
5
5
5
5

0.16
0.54
0.38
0.54
0.56

Cut

0.97

5

0.86

Uncut

0.91

5

0.49

Cut
Uncut
Cut
Uncut

0.98
0.84
0.9
0.93

5
5
5
5

0.91
0.18
0.39
0.61

Cut

0.87

5

0.28

Uncut

0.82

5

0.12

Cut

0.9

5

0.43

Mann-Whitney U Test Results
Test Statistic: Degrees of Freedom: P-value:

Tail Beating
Tail Flashing
Flaring Gills
Extending Gill Membrane
Raising Dorsal Fin
Lowering Head
Darting Toward Opponent
Nipping at Opponent

-0.94
1.16
-1.15
-1.15
-0.63
-0.11
-1.19
0.31

Table 3. Statistical results from the Mann-Whitney
U test on the number of times aggressive behaviors
were displayed.
Mann-Whitney U Test Results
Aggressive Display:
Test Statistic: Degrees of Freedom: P-value:
Tail Beating
-0.73
10
0.55
Tail Flashing
-0.94
10
0.42
Flaring Gills
-0.52
10
0.69
Extending Gill Membrane
-0.52
10
0.69
Raising Dorsal Fin
2.61
10
0.008
Lowering Head
2.20
10
0.03
Darting Toward Opponent
-1.26
10
0.22

10
10
10
10
10
10
10
10

0.42
0.31
0.31
0.31
0.55
1.00
0.31
0.84

Table 4. Statistical results from the Mann-Whitney
U test on the amount of time aggressive behaviors
were displayed.

Figure 10. Average number of times tail beating. Error
bars show standard error. t=-0.94, df=10, p=0.42

Figure 11. Average amount of time tail beating. Error
bars show standard error. t=-0.73, df=10, p=0.55

Figure 12. Average number of times tail flashing. Error
bars show standard error. t=1.16, df=10, p=0.31

Figure 15. Average amount of time flaring gills. Error
bars show standard error. t=-0.52, df=10, p=0.69

Figure 14. Average number of times flaring gills. Error
bars show standard error. t=-1.15, df=10, p=0.31

Figure 13. Average amount of time tail beating. Error
bars show standard error. t=-0.94, df=10, p=0.42

Figure 16. Average number of times extending the gill membrane.
Error bars show standard error. t=-1.15, df=10, p=0.31

Figure 18. Average number of times raising the dorsal fin. Error
bars show standard error. t=-0.63, df=10, p=0.55

Figure 17. Average amount of time extending gill membrane.
Error bars show standard error. t=-0.52, df=10, p=0.69

Figure 19. Average amount of time raising the dorsal fin. Error
bars show standard error. t=2.61, df=10, p=0.008

Figure 20. Average number of times lowering head.
Error bars show standard error. t=-0.11, df=10, p=1.00

Figure 21. Average amount of time lowering head. Error
bars show standard error. t=2.20, df=10, p=0.03

Figure 22. Average number of times darting toward opponent.
Error bars show standard error. t=-1.19, df=10, p=0.31

Discussion:

Figure 23. Average amount of time darting toward opponent.
Error bars show standard error. t=-1.26, df=10, p=0.22

Figure 24. Average number of times nipping at
opponent. Error bars show standard error. t=0.31,
df=10, p=0.84

The lack of statistical significance in male Betta splendens with cut tails
compared to male Betta splendens with uncut tails in both the amount and number of
times aggressive behaviors were displayed suggests that tail regeneration generally does
not affect their aggressive behavior towards other males.
This study shows that the regenerated tissue is functionally normal. Additionally,
this information is consistent with findings from zebrafish tail amputation research,
suggesting that complex fin regeneration is very similar to simple fin regeneration.
Moreover, this lack of significance on aggressive behavioral displays indicates that the
Betta splendens must not have experienced enough psychological harm to change their
behavior due to the amputation.
Tail amputation was thought to affect either the number of times or the amount of
time aggressive behavior was displayed in male Betta splendens since many of their
aggressive behaviors include their tail. Amputation would decrease the size of their tail,
generally even when regrown, and the size they could make themselves look towards
other opponents, which would theoretically make them back off from fighting other male
Betta splendens. However, this hypothesis suggests that Betta splendens are aware of the
size of their tail, which is yet to be determined.
The two behaviors, raising the dorsal fin and lowering the head, that showed a
statistically significant higher display in males with cut tails suggests male Betta
splendens are in some way aware of their tail size, since they resort more frequently to
aggressive behaviors that do not depend on their tail. They may have used longer dorsal
fin and head-lowering displays to make up for that new weakness. The data set used in
this experiment was the smallest possible though, so the results could change drastically
if a larger sample size is used. Future studies should conduct the same experiment with a
larger sample size to verify the results.
The lack of statistical significance found also suggests that wild Betta splendens
would still fight to the death to increase their own fitness, despite a substantial tail injury
that may make them look weaker or smaller when compared to their uninjured opponent.
This evidence suggests that the cost of tail regeneration is less than the cost of not
displaying aggression. If the male Betta splendens do not display aggression, they forgo
their chances of passing on their genes to the next generation, which significantly lowers
their fitness. Also, multiple sources describe that behavioral displays are a way for the
males to “resolve the conflict without costly escalated fighting” (Castro, et al., 2006;
Caryl, 1979; Maan et al., 2001; Neat et al., 1998; Zahavi, 1977). Thus, it makes sense that
males who experienced regeneration still display aggressive behaviors since they are less
costly than physically fighting for their fitness. However, since tail regeneration was not
examined in the presence of a female Betta splendens, no conclusions can be made about
how this might affect their fitness in terms of finding a partner to mate with. Future
studies could focus on how if length after amputation changes how aggressive Betta
splendens are towards their opponent, or even how tail amputation might affect a male
Betta splendens fitness.

Unfortunately, epigenetic data could not be tested to see if the tail amputation
affected the epigenetic in the males with cut tails. Thus, it is difficult to conclude how the
Betta splendens may have been physically stressed due to this experience. If epigenetic
changes occurred, this would indicate that there are long-term changes in the gene
expression associated with amputation and regeneration.
As a result, Betta splendens could act as a model organism, if need be, since they
show more ability than zebrafish in recognizing other males and forming social
hierarchies (Forsatkar, et al., 2017; Jameson, et al., 1999). Their development could be
further studied to find how simulated traumatic injuries may trigger regeneration and
how, when applied in humans, it could affect their epigenetics and behavior in the future.

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APPENDIX:

Average Number of Occurances of Various Behaviors
Behavior

Uncut vs. Cut Average Number of Occurances

Std. Dev. Std. Error Normal Data?

Tail Beating
Uncut tails
Cut tails

11.33
7.00

10.78
4.06

4.82
1.82

Yes
Yes

Uncut tails

3.60

2.07

0.93

Yes

Cut tails

7.00

5.24

2.35

Yes

Uncut tails
Cut tails

25.40
18.40

16.10
22.85

7.20
10.22

Yes
No

Uncut tails

25.20

14.34

6.41

Yes

Cut tails

16.60

19.83

8.87

No

Uncut tails
Cut tails

15.80
9.20

12.76
3.83

5.70
1.71

Yes
Yes

Uncut tails
Cut tails

13.20
12.20

8.04
6.53

3.60
2.92

Yes
Yes

Uncut tails
Cut tails

9.80
2.40

10.08
1.34

4.51
0.60

No
Yes

Uncut tails
Cut tails

8.20
19.80

5.40
23.83

2.42
10.66

Yes
No

Tail Flashing

Flaring Gills

Extending Membrane

Raising Dorsal Fin

Lowering Head

Darting Toward Opponent

Nipping at Opponent

Average Length of Time in Seconds that Various Behaviors Occur
Behavior
Tail Beating

Uncut vs. Cut Average Length of Time in Seconds

Std. Dev. Std. Error Normal Data?

Uncut tails
Cut tails

63.00
27.80

52.55
21.78

23.50
9.74

Yes
Yes

Uncut tails

222.40

82.48

36.89

Yes

Cut tails

153.60

89.75

40.14

Yes

Uncut tails

135.40

62.95

28.15

Yes

Cut tails

116.40

87.85

39.29

Yes

Uncut tails

147.40

66.09

29.56

Yes

Cut tails

122.20

85.19

38.10

Yes

Uncut tails
Cut tails

15.80
150.60

12.76
57.87

5.70
25.88

Yes
Yes

Uncut tails
Cut tails

13.20
60.00

8.04
44.12

3.60
19.73

Yes
Yes

Uncut tails
Cut tails

29.20
8.00

31.77
5.43

14.21
2.43

Yes
Yes

Tail Flashing

Flaring Gills

Extending Membrane

Raising Dorsal Fin

Lowering Head

Darting Toward Opponent