GENDER BIAS IN BYSTANDER EFFECTS FOR AGGRESSIVE DISPLAYS IN BETTA SPLENDENS

Heather Schofield, Althia Rickard, Malisa Rai

April 20th, 2018

Bio 345 – Animal Behavior

Abstract:

 Betta splendens are model organisms for understanding aggressive behavior in aquatic species. Aggressive displays have been well documented and include the opercula display and hitting with the tail. These behaviors have evolutionary contexts that have created differences in the frequencies that these behaviors are displayed in the presence of various audience types. Male betta fish were observed without bystanders and with different gendered bystanders to determine differences between these displays. There were significantly less gill flares in the presence of a female bystander. The presence of a male bystander saw significantly less tail flicking. The data suggested that gill flaring is typically more common in the presence of other males whereas tail beating is more closely associated with the presence of a female bystander. It is believed that tail beating evolved from an aggressive display to a courtship display due to chase away selection.

Introduction:

Communication between animals occurs in a variety of forms, such as auditory, electrical, or visual signals. The use of signals allows animals to send information to one another and modify behaviors based upon the understanding of those signals[7]. A few common reasons that animals interact are for mate attraction, territory or predator defense, and social integration. Aggressive behavior in animals is common for defensive mechanisms and acquiring resources [6]. Sometimes these signals accurately depict an animal’s true size and ability which is called honest signaling. When the signals are misleading this is described as dishonest signaling, however, evolution tends to favor honest signaling [8]. Gill flaring, also called the opercula display, is an honest signal that male Betta splendens use to intimidate an opponent and suggest fighting ability and strength. This behavior of gill flaring is energetically costly because the display prevents the fish from using the gills to obtain oxygen which requires stamina [10]. Traits that are used for communication are evolutionarily and historically limited based upon the phylogeny of the species. Only a pre-existing trait such as physiological abilities and behaviors can become an evolutionary adaptation. These become incorporated into the larger population if they increase the overall fitness of the individuals with that trait [8].

Another aspect of communication is eavesdropping where an individual receives information about another from a signal that was not intended for it. This is seen in a variety of animals and often the eavesdropper behavior toward the signaler is influenced by the signal that was intercepted. An example of eavesdropping is when snakes use the mating calls of frogs to find their prey [9]. Other studies on eavesdropping as a communication mechanism have suggested that male Betta splendens alter their aggression displays depending on the context of the situation. It was found that the sex of a bystander, among other contextual elements, has significant effects on the display behavior of Betta splendens. The males showed different levels of aggression depending on the presence or absence of an audience, the gender of the audience, their own reproductive state, and the amount of resources held [2].

Siamese fighting fish, Betta splendens, are a model organism for observing fish aggression. Typical aggressive behaviors that have been previously documented on Betta splendens include, but are not limited to, gill flaring, fin spreading, tail beating, and biting. Physical displays such as tail beating and biting only occur when the benefits of territoriality outweigh the costs of potential harm of fighting [4].Tail beating has been more closely associated with courtship rituals and therefore are thought to occur more often in the presence of female Betta Splendens. The gill flaring behavior was shown to be greatest in the presence Betta splendens who were males [3]. This suggests that there may in fact be a gender-based bystander effect on the aggression displays in male Betta splendens.

This study aimed to investigate aggressive behaviors in male Betta Splendens to determine the response to the presence and absence of different gendered bystanders. This was done to shed light on the evolutionary and historical outcomes of specific aggressive behaviors in Betta splendens. We hypothesize that Betta splendens will have differential aggressive displays that are dependent on the gender of the bystander. We predicted that if a male Betta Splendens is placed in a tank with a mirror, it would display more gill flaring than tail flicking when there is a male Betta Splendens bystander. We also predicted that if a male Betta Splendens is placed in a tank with a mirror, it would display more tail beating than gill flaring when there is a female Betta Splendens bystander.

Methods:

Study Species:

Five mature male crowntail Betta splendens and one mature elephant ear female from the local Petco were purchased. The males varied in size, shape, and color to be representative of the variation observed in male betta fish. They are naturally found in freshwater ponds of Southeast Asia. Captive-bred and wild males both exhibit strong and stereotyped aggression in defending their territories against intruding male conspecifics.  Each male fish was assigned a number for later identification.

Housing and Care:

 All fish were kept in a small isolated glass fish bowl with about 700mL of lukewarm tap water and black pebbles for resting on. These fishbowls were initially housed on the fourth floor of the KSC Putnam Science Center in the greenhouse and then later moved to the greenhouse office for a cooler environment. The water in all fish bowls and the experimental tank was treated with  API  Splendid Betta Complete Water Conditioner from Petco to prepare the  tanks for suitable living conditions [4].  The conditioner is used to remove chemicals from the treated tap water because these chemicals are toxic to fish [5]. Fish bowls remained segregated to prevent interactions. Fish were fed generic betta fish food from Petco daily. Fish bowls were cleaned every other day or more often if needed, all water was treated with the API conditioner before fish were placed into the water. The water was maintained at about neutral pH and the water temperature was held at room temperature.

The female betta perished the day after purchase and was replaced with a similar elephant ear female at Petco. Male #1 perished after the self-trials and was removed from the experimental data set. He was not replaced and the trials continued with only Male #2, #3, #4, #5, and the female. It was believed that the heat of the greenhouse caused these fatalities and the fish were moved inside to the greenhouse office to remain cooler.

Experimental Tank Set-up:

A half-gallon tank with a divider was filled with tap water and treated with conditioner. One male betta was placed into a plastic bag and allowed to acclimate to side A of the experimental tank. An opaque cover was placed over the divider to prevent interactions prior to the trials (Figure 1). A male or female betta acclimated to side B, the bystander side. After each trial the tank was emptied and cleaned and the water was treated with API conditioner.

Figure 1: Experimental half-gallon tank with a divider. Side A will contain the male Betta that will be exposed to the mirror. Location of the mirror is shown on the back wall of side A.  Side B will be used to house the bystander that will be male or female.

Experimental Design:

A small mirror was attached to the wall of the experimental tank on side A. Male betta #2 was allowed to acclimate to the experimental tank on side A. Then for five minutes, male aggressive displays of gill flaring and tail flicking were counted and recorded. Three trials of solitary displaying, trials without bystanders, were done for each fish. These trials served as a control to compare solitary versus bystander behaviors.

Male #3 was placed on side B of the experimental tank and was allowed to adjust to the new environment. The opaque cover for the divider prevented interactions. Once both fish in side A and B, separated by the opaque divider, were acclimated to the water, the trial began. The opaque divider was removed so that the bystander was visible. For five minutes, the number of tail flicking and gill flaring that betta #2 displayed were recorded.

After the five minutes the fish were placed in their respective fish bowls for acclimation while the experimental tank was cleaned. Male #2 was put back into side A to be tested with male #4 and this was repeated for male #5 and the female betta. All five males were tested in side A for solitary mirror displays and bystander effects. Each male was tested with the female three times. Each set of male trials (#2, 3, 4, 5) was a replicate experiment.    

Focal Betta Behaviors:

This study aimed to investigate the differences in gill flaring and tail beating behaviors of male betta fish when exposed to either a male or female bystander betta fish. The behaviors are described in Table 1. These behaviors were chosen because they are easily spotted and have been described in published papers on this species.

Table 1: Descriptions of aggressive betta fish behaviors derived from primary literature search [3] and observations.

Behavior: Description:
Gill Flaring Opercula display, extension of gills
Tail Beating Using the tail to hit or attempt to hit an opponent or object

 

Statistical Analysis 

            The averages of each trial condition (self, male bystander, female bystander) were calculated with standard deviations and standard errors using Microsoft Excel. A One-Way ANOVA with post hoc comparisons were done for gill flares and for tail beats using an online calculator. The post hoc comparisons were used to determine the significance of the data between the different trial conditions. The average number of gill flares for each trial condition (self, male bystander, female bystander) were calculated and compared. The average tail beats for each trial condition were calculated and compared. The significance of each type of trial condition was calculated for each behavior (gill flaring or tail beating).

Results:

            Gill flaring behavior appeared to decrease in frequency in the presence of a female bystander. The statistical analysis indicated that there were significant differences between the average number of the two aggressive displays with a male versus a female bystander for the three different trial conditions. The results showed that there were significantly less gill flares in the trials that had the female bystander (P= 0.0023, F=6.762, Figure 2) as compared to a male bystander and the self-trials. Gill flares for the self-trials and male bystander trials were not significantly different suggesting that it was the presence of the female creating the differences. The different letters above the data bars indicate if the data sets are significantly different.

 

Figure 2: Average number of Gill Flares exhibited by the focal male Betta during the five- minute trials. For the three trial conditions the F value = 6.7622 and P-value = 0.0023. A male Betta splendens was in the experimental tank with a mirror on side A and observed for five minutes displaying to a mirror with either nothing, a male bystander, or a female bystander in side B of the experimental tank. The number of gill flares in the female bystander trial condition was significantly less than the other two trial conditions.

The self-trials and female bystander trials appeared to have similar means for tail beating when displayed graphically. There was a significant difference between the number of tail beats in the presence of a male versus a female bystander (P=0.0015, F=7.3241, Figure 3). There were greater tail beating events in the presence of the female bystander and the self-trials when compared to the male bystander trials. This data showed that in the presence of male bystanders there were a greater number of gill flares and that there were more tail beatings in the presence of the female bystander.

Figure 3: Average number of Tail Beats exhibited by the focal male Betta during the five-minute trials. F-value = 7.3241. P-value = 0.0015. A male Betta splendens was in the experimental tank with a mirror on side A and observed for five minutes displaying to a mirror with either nothing, a male bystander, or a female bystander in side B of the experimental tank. Male bystander trial conditions had significantly less tail beating events than the other two trial conditions.

Discussion:

Aggressive behaviors are typically seen in response to territory defense and mate acquisition. These behaviors have evolutionary contexts that has been selected for over thousands of years. The Betta splendens is a classic fish pet that has been studied extensively for their aggressive behaviors. It has been proposed that there is gender based bystander effects that determine the frequency that specific aggressive behaviors are displayed due to the audience effect [1]. This study aimed to investigate whether there was a gender bias for gill flaring and tail beating in male Betta splendens. Various colored and sized males were observed displaying to a mirror without a bystander, with a male bystander, and with a female bystander.

It had been hypothesized that there would be greater gill flare displays in the presence of male bystander whereas a female bystander would induce more tail beatings. The experimental trials supported this hypothesis. The male bystander trials displayed significantly more gill flares compared to the female bystander suggesting that gill flaring is typically used as an aggressive defense against invaders. The female bystander trials showed significantly more tail beatings suggesting that this behavior has both aggressive and mating evolutionary contexts. This suggests that there are differences in aggressive displays due to the presence of different gendered bystanders. Future experiments or observations could look at how or if closely related species exhibit the same aggressive behaviors based upon the sex of bystanders or if this is unique to Betta splendens.

References:

[1] Claire Doutrelant, Peter K. McGregor, Rui F. Oliveira; The effect of an audience on intrasexual communication in male Siamese fighting fish, Betta splendens, Behavioral Ecology, Volume 12, Issue 3, 1 May 2001, Pages 283–286, https://doi.org/10.1093/beheco/12.3.283

[2] Dzieweczynski, Teresa L., et al. “Audience Effect Is Context Dependent in Siamese Fighting Fish, Betta Splendens .” OUP Academic, Oxford University Press, 29 Sept. 2005, academic.oup.com/beheco/article/16/6/1025/216546.

[3] Dzieweczynski, Teresa, et al. “Opponent Familiarity Influences the Audience Effect in Male–Male Interactions in Siamese Fighting Fish.” Animal Behaviour, Academic Press, 15 Mar. 2012, www.sciencedirect.com/science/article/pii/S0003347212000905.

[4] Egar M. J, Lynn E. S, Ramenofsky. M, Sperry S. T, Walker G. B. (2007). “Fish on Prozac: a simple, noninvasive physiology laboratory investigating the mechanisms of aggressive behavior in Betta Splendens.” American physiological society. Retrieved from http://www.physiology.org/doi/10.1152/advan.00024.2007

[5] Mohrman, Eric. “What Does Conditioner Do for an Aquarium?” Pets, The Nest, 2007, pets.thenest.com/conditioner-aquarium-11906.html.

[6] Romano, Donato, et al. “Multiple Cues Produced by a Robotic Fish Modulate Aggressive Behaviour in Siamese Fighting Fishes.” Scientific Reports, Nature Publishing Group UK, 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5498610/#annotations:OnbKwBeQEeioFRO-O4fMiA.

[7] Rosenthal, Gil G. “Spatiotemporal Dimensions of Visual Signals in Animal Communication.”Annual Review of Ecology, Evolution, and Systematics, vol. 38, 2007, pp. 155–178. Illiad, doi:10.1146/annurev.ecolsys.38.091206.095.

[8] Schofield , Heather. “Communication Between Animals.” Schofield Investigations, KSC Open , 1 Mar. 2018. schofieldinvestigations.kscopen.org/schofield-courses-00345/kscanimbehav/communication-between-animals/.

[9] Strauss, Amy. “Eavesdropping in the Animal Kingdom: Sneaky Creatures Just Trying to Get Ahead.” Thats Life Science, 5 Sept. 2017, thatslifesci.com.s3-website-us-east-1.amazonaws.com/2017-09-05-Eavesdropping-in-the-Animal-Kingdom-Sneaky-Creatures-Just-Trying-to-Get-Ahead-AStrauss/.

[10] Verbeek, Peter, et al. “Differences in Aggression between Wild-Type and Domesticated Fighting Fish Are Context Dependent.” Animal Behaviour, vol. 73, no. 1, 2007, pp. 75–83., doi:10.1016/j.anbehav.2006.03.012.

 

 

 

 

 

 

 

 

 

Please follow and like:
error

Progress Update #5

“Male #3 in the experimenal tank” by H. Schofield (CC by 2.0)

Data was collected on 4/5/18 and 4/8/18. The trials were completed and the averages of the trials were calculated. The fish were slightly more active the second. The female stayed close to the divider and interacted with the males often.

Data Collection: The Completion of the Trials 

Side A: Focal Fish Side B: Bystander Avg. Gill Flares Avg. Tail Beats
Male #2 Nothing-self trial 40.33 15.33
Male #2 Male betta 13.167 4.417
Male #2 Female betta 1.33 5
Male #3 Nothing- self trial 26 8.667
Male #3 Male betta 7 2
Male #3 Female betta 6.667 11.667
Male #4 Nothing- self trial 8.33 3.33
Make #4 Male betta 15.889 3.778
Male #4 Female betta 8.33 9.33
Male #5 Nothing- self trial 3 1
Male #5 Male betta 11.22 3.778
Male #5 Female betta 1.33 6.667

Table 1: Averages from the self trials and crossed trials.. 

Please follow and like:
error

Progress Update #4

“Male #3 gill flaring to the Female Betta splendens” by H. Schofield (CC by 2.0)

This data was collected 4/3/18 and 4/4/18. More male/male trials were completed and most of the male/female trials were completed. Each pair was tested 3 times for a total of 15 minutes under inspection. The experimental tank was used for all of these trials. Experiment side A housed the focal male and contained the mirror. Experiment side B housed the bystander. The pebbles at the bottom of the experiment tank were removed due to interference with the divider.

The female fish was placed on experiment side B for Male #2, #3, and #5. There appears to be a possible reversal in the behavior that the male preferentially displays. There were low levels of activity in all fish during both days of these trials. The female attempted to interact with each male that she was placed with. she displayed her tail fin and swam the length of the divider often. She faced toward the divider and often followed the male if he swam along the divider of the tank. When Male #3 was in experiment side A and the female in experiment side B, when he gill flared at her, she responded with a gill flare. Although it was not as impressive, she was able to extend her gills partially to puff them out.

Data Collection: The Introduction of the Female Betta splendens 

Trial # Experiment Side A Experiment Side B Gill Flares Tail Beats
1 Male #2 Male #3 31 5
2 Male #2 Male #3 35 10
3 Male #2 Male #3 37 5
Average: 34.33 6.66
1 Male #2 Male #4 3 0
2 Male #2 Male #4 12 5
3 Male #2 Male #4 7 4
Average: 7.33 3
1 Male #2 Male #5 6 2
2 Male #2 Male #5 13 4
3 Male #2 Male #5 10 3
Average: 9.667 3
1 Male #2 Female 1 4
2 Male #2 Female 1 5
3 Male #2 Female 2 6
Average: 1.33 5
1 Male #3 Female 6 10
2 Male #3 Female 5 13
3 Male #3 Female 9 12
Average: 6.66 11.66
1 Male #4 Male #5 14 1
2 Male #4 Male #5 21 2
3 Male #4 Male #5 16 1
Average: 17 1.33
1 Male #5 Female 1 5
2 Male #5 Female 2 8
3 Male #5 Female 1 7
Average: 1.33 6.66

Table 1: Crossed Trials with Betta splendens. Counts of gill flares and tail beats per five minute trial. Averages of each pair were calculated. 

Please follow and like:
error

Progress Update #3

April 1st, 2018

3 PM

The bystander trials began today. Using two pairs of fish six trials were completed. Male #2 and Male #4 were placed into experiment side A with the mirror, in each pair, these two fish were the focus of counting gill flares and tail beats in each five minute trial. During the trial with Male #2 and #3 there was a lot of interaction between the two fish as well as the interaction that Male #2 had with the mirror. Male #3 interacted with Male #2 as well by gill flaring, tail beating, fin spreading, and attempting to bite the divider. Male #4 was more interactive with the mirror than Male #5 who was in experiment side B. Male #5 did not display any aggressive behaviors. He approached the divider and watched Male #4 a cpuple of times but remained on the far side of the tank for the majority of the time. After the trials were completed all fish bowls and the experiment tank were cleaned.

“Male #2 and Male #3 bystander trial” by H. Schofield (CC by 2.0)

Today’s Trials: The Beginging of the Bystanders 

Trial # Experiment Side A Experiment Side B Gill Flares Tail Beats
1 Male #2 Male #3 31 5
2 Male #2 Male #3 35 10
3 Male #2 Male #3 37 5
Average: 34.33 6.66
1 Male #4 Male #5 14 1
2 Male #4 Male #5 21 2
3 Male #4 Male #5 16 1
Average: 17 1.33

Table 1: Bystander trials. Experiment side A  contained a mirror, there was a clear divider between the two sides. Trials were each five miniutes in length. 

Please follow and like:
error

Progress Update #2

3/29/18

12 PM

All fish were active and swimming in their bowls. There was aa small amount of  leftover food from yesterday in all but male #5’s fishbowl. Tanks are a still clear. The self trials for the males will be continued today during the regularly scheduled class period (12-1:45 PM). All tanks were cleaned after the trials were completed.

“Trial #3 with Male #5” by H. Schofield (CC by 2.0)

Important Terms to Remember:

Gill Flaring: an opcuoar display with the extension of the gills. This is honest signaling because the fish is unable to breathe during this display and this indicates the fish’s fighting ability and strength.

Tail Flicking/Beating: when a betta attempts to hit the oppoent with the large colorful tail. It is thought to be associated with courtship rituals and is hypothesized to occur more frequently when there is a female bystander.

Today’s Data and the Completion of the Male Self Trials:

Fish and Trial #
# of Gill Flares
# of Tail Beats
Male #2 Self Tial
1 46 23
2 50 10
3 25 13
Average: 40.33333333 15.33333333
Male #3 Self Tial
1 31 7
2 24 9
3 23 10
Average: 26 8.666666667
Male #4 Self Trial
1 14 5
2 6 2
3 5 3
Average: 8.333333333 3.333333333
Male #5 Self Trial
1 0 0
2 3 1
3 6 2
Average: 3 1

Table 1: Self Trials for Male Betta Fish Aggression Displays for three five minute trials with a mirror. 

 

 

Please follow and like:
error

Progess Update #1

3/28/18

3pm

Upon coming into the greenhouse, the team discovered that Male Betta #1 had died since yesterday’s feeding. He will be removed from the experiment but not replaced. The four remaining males will be used for data collection.

Previously the female that had been obtained had also died and was replaced over the weekend. The new female is still alive.

“Replacement female elephant ear betta fish” by H. Schofield (CC by 2.0)

The self trials with the male betta fish began today. For the begining of the experiment Betta #2 remained at the surface of the experimental tank. It took about five minutes of being within the tank to encounter the mirror. We lowered the water levels so that he was forced to stay within sight of the mirror. After several interactions with the mirror the actual trial began. For five minutes the number of tail flicking and gill flaring were counted. Other interesting behaviors of Male #2 were his attempts to swim behind the mirror. The group discussed getting a larger mirror that would cover the entire side of the tank so that there was a higher liklihood of the fish viewing itself in the mirror.  Male #3 attempted to bite the mirror aggressively many times and we also saw him spread his tail quite frequently to display to his refleciton. Male #4 was not very interactive with the mirror. He remained on the other side of the tank and only occassionally approached and interacted with the reflection. Male #4 is the smallest male and he is mostly white. Male #5 did not interact with the mirror at all. We believe this is due to heat exhaustion from being housed in the greenhouse. We have moved all fish into a cooler room to prevent further death in the specimen and to hopefully increase activity levels in the fish.

Today’s Data Collection: 

Male #2:  

  • Trial 1:
    • 46 gill flares
    • 23 tail flicks
  • Trial 2:
    • 50 gill flares
    • 10 tail flicks

Male #3:

  • Trial 1:
    • 31 gill flares
    • 5 tail flicks

Male #4:

  • Trial 1:
    • 14 gill flares
    • 5 tail flicks

Male #5

  • no interaction with the mirror was observed in the five minute trial.

Further data collection and progress updates will follow. 

Please follow and like:
error

Betta Fish Experimental Set-up

“Fish cubbies” by H. Schofield (CC by 2.0)

3/22/18

The team added black rocks, tap water, and API Tap Water Conditioner to six small glass bowls. These bowls were labled as Male #1 up to Male #5, and one bowl was labelled Female. All six bowls were placed on a table in the greenhouse on the top floor of the science center and separated by folders.  The experiemtnal tank that was bought at petco has two chanbers separated by a divider with a removable black part to allow visibility between both sides. This tank was also filled with black rocks, tap water, and API Tap Water Conditioner. Five Crowntail males and one Elephant Ear female were purchased from Petco and placed into the prepared fish bowls. After the adjustment period, the fish were fed.

“Male #1” by H. Schofield (CC by 2.0)

Male #1: large dark blue body, turquoise pectoral fins, the caudal fin is both blues. The pelvic fins are red. The Dorsal fins are dark blue.

“Male #2” by H. Schofield (CC by 2.0)

Male #2:  Dark blue body, top of back is green blue, pectoral fins are  white, caudal fin is turquoise and dark blue with white tips, the pelvic fins are white, the dorsal fins are turquoise with red tips.

“Male #3” by H. Schofield (CC by 2.0)

Male #3:  The body is white with some red patches,  the pectoral fins are patchy with red, white, and blue. The the caudal fin is turquoise and red, the pelvic fins are blood red, and the dorsal fins are light blue and white at the base and red at the tips.

“Male #4” by H. Schofield (CC by 2.0)

Male #4:  This fish is all white.

“Male #5” by H. Schofield (CC by 2.0)

Male #5:  This male is all blood red.

“Female Betta” by H. Schofield (CC by 2.0)

Female: She has a reddish head, pale blue body, and all her fins are white with a light blue tint.

Please follow and like:
error

Project Proposal: Gender Bias in Bystander Effects for Aggressive Displays in Betta splendens

¨Betta¨by Neil Mullins (CC BY  2.0)

Co-authors: Malisa Rai and Althia Rickard

Background:

Communication between animals occurs in a variety of forms, such as auditory, electrical, or visual signals. The use of signals allows animals to send information to one another and modify behaviors based upon the understanding of those signals[7]. A few common reasons that animals interact are for mate attraction, territory and predator defense, and social integration. Aggressive behavior in animals is common for defensive mechanisms and acquiring resources [6]. Sometimes these signals accurately depict an animal’s true size and ability which is called honest signaling. When the signals are misleading this is described as dishonest signaling, however, evolutions tend to favor honest signaling. Gill flaring also called the opercular display, is an honest signal that male Betta splendens use to intimidate an opponent and suggest fighting ability and strength. This behavior of gill flaring is energetically costly because the display prevents the fish from using the gills to obtain oxygen which requires stamina [9]. Traits that are used within communication are evolutionarily and historically limited based upon the phylogeny of the species. Only a pre-existing trait such as physiological abilities and behaviors can become an evolutionary adaptation. These become incorporated into the larger population if they increase the overall fitness of the individuals with that trait [8]. Another aspect of communication is eavesdropping where an individual is able to receive information about another from a signal that was not intended for it. This is seen in a variety of animals and often the eavesdropper behavior toward the signaler is influenced by the signal that was intercepted. Studies have suggested that male Betta splendens alter their aggression displays depending on the context of the situation. It was found that the sex of a bystander, among other contextual elements, has significant effects on the display behavior of Betta splendens. The males showed different levels of aggression depending on the presence or absence of an audience, the gender of the audience, their own reproductive state, and the amount of resources held [1].

Siamese fighting fish, Betta splendens are a model organism for monitoring fish aggression. Typical aggressive behaviors that have been previously documented on Betta splendens include, but are not limited to, gill flaring, fin spreading, tail beating, and biting. Physical displays such as tail beating and biting only occur when the benefits of territoriality outweigh the costs of potential harm of fighting [3].Tail beating has been more closely associated with courtship rituals and therefore occur more often in the presence of female Betta Splendens whereas gill flaring was shown to be greatest in the presence of unknown Betta splendens who were males [2]. This suggests that there may in fact be a gender-based bystander effect on the aggression displays in male Betta splendens.

 

Objectives and Hypothesis:

This study aims to investigate the different aggressive behaviors in male Betta Splendens to determine the  response to the presence and absence of bystanders. We wondered if  the gender of a bystander affected aggressive displays in male Betta splendens .

We hypothesize that Betta splendens will display different aggressive behaviors to a mirror in the presence of different gendered bystanders.

  • We predict that if a male Betta Splendens is allowed to view themselves in a mirror they will display more gill flaring than tail flicking when there is a male Betta Splendens bystander. 
  • We predict that if a male Betta Splendens is allowed to view themselves in a mirror they will display more tail flicking than gill flaring when there is a female Betta Splendens bystander. 

 

Methods:

Study Species:

We will obtain five mature male Betta splendens and one mature female from the local pet store, they will vary in size, shape, and color to be representative of the variation observed in male betta fish. They are naturally found in freshwater ponds of Southeast Asia. Captive-bred and wild males both exhibit strong and stereotyped aggression in defending their territories against intruding male conspecifics [3].  The 5 males and 1 female Betta splendens will be kept in isolated 1 gallon glass tanks with black pebbles for resting on. They will not be able to view each other. Tap water will be used in all fish tanks and will be conditioned with API  Splendid Betta Complete Water Conditioner from Petco to prepare the  tanks for suitable living conditions.  The conditioner is used  to remove chemicals from the treated tap water because these chemicals are toxic to fish [4]The water will be maintained at about neutral pH and the water temperature will be held at room temperature. They will be fed daily using a generic betta fish product found at the pet store. Each male fish will be given a number so that they can be later identified. Tanks will be cleaned once weekly. 

Experimental Tank Set-up:

A 5 gallon tank with a divider will be filled with tap water and treated with start right. One male betta will be placed into a plastic bag and allowed to acclimate to side A of the experimental tank. An opaque cover will be placed over the divider to prevent interactions prior to the trials. A male or female betta will be allowed to acclimate to side B, the bystander side. After each trial the tank will be emptied and cleaned. The first male betta will remain as the mirror exposed in side A until it has cycled through all other four males and the female. This will occur for all five of the males, each male serves as a replicate.

Figure 1: Experimental 5 gallon tank with a divider. Side A will contain the male Betta that will be exposed to the mirror. Location of the mirror is indicated by the yellow mark in side A.  Side B will be used to house the bystander.

Experimental Design:

A small mirror will be attached to the wall of tank A. Male betta #1 will be allowed to acclimate to the experimental tank on side A. Then for five minutes, male aggressive displays of gill flaring and tail flicking will be counted and recorded. Three trials of solitary displaying will be done for each fish. This will serve as a control to compare solitary versus bystander behaviors.

Male #2 will be allowed to acclimate on side B of the experimental tank. The opaque cover for the divider will prevent interactions. Once both fish in side A and B, separated by the divider with an opaque diver, are acclimated to the water the trial will begin. The opaque divider will be removed so that the bystander is now visible. For five minutes, the number of tail flicking and gill flaring that betta #1 displays will be recorded.

After the five minutes the fish will be placed in their respective 1-gallon tanks for acclimation while the experimental tank is cleansed. Male #1 will be put back into side A to be tested with male #3 and this will be repeated for male #4, #5, and the female betta. All five males will be tested in side A for solitary mirror displays and bystander effects. Each male will be tested with the female three times. 

Focal Betta Behaviors:

This study aims to investigate the differences in gill flaring and tail flicking of male betta fish when exposed to either a male or female bystander betta fish.

Behavior: Description:
Gill Flaring Opercular display, extension of gills
Tail Flicking Using the tail to hit or attempt to hit an opponent

Table 1: Descriptions of  aggressive betta fish behaviors derived from primary literature search [2].

Statistical Analysis  

A two tailed paired T-test will be used to analyze the results. The number of times that each male betta spends gill flaring or tail beating when alone will be compared to the number of times he does those behaviors in the presence of different gendered bystanders.

Male Betta ID number # of gill flaring, no bystander # of  gill flaring, male bystander # of gill flaring, female bystander # of tail beating, no bystander # of tail beating, male bystander
# of tail beating, female bystander
1
2
3
4
5

Table 2: Sample data collection table.

 

References:

[1] Dzieweczynski, Teresa L., et al. “Audience Effect Is Context Dependent in Siamese Fighting Fish, Betta Splendens .” OUP Academic, Oxford University Press, 29 Sept. 2005, academic.oup.com/beheco/article/16/6/1025/216546.

[2] Dzieweczynski, Teresa, et al. “Opponent Familiarity Influences the Audience Effect in Male–Male Interactions in Siamese Fighting Fish.” Animal Behaviour, Academic Press, 15 Mar. 2012, www.sciencedirect.com/science/article/pii/S0003347212000905.

[3] Egar M. J, Lynn E. S, Ramenofsky. M, Sperry S. T, Walker G. B. (2007). “Fish on Prozac: a simple, noninvasive physiology laboratory investigating the mechanisms of aggressive behavior in Betta Splendens.” American physiological society. Retrieved from http://www.physiology.org/doi/10.1152/advan.00024.2007

[4] Mohrman, Eric. “What Does Conditioner Do for an Aquarium?” Pets, The Nest, 2007, pets.thenest.com/conditioner-aquarium-11906.html.

[6] Romano, Donato, et al. “Multiple Cues Produced by a Robotic Fish Modulate Aggressive Behaviour in Siamese Fighting Fishes.” Scientific Reports, Nature Publishing Group UK, 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5498610/#annotations:OnbKwBeQEeioFRO-O4fMiA.

[7] Rosenthal, Gil G. “Spatiotemporal Dimensions of Visual Signals in Animal Communication.”Annual Review of Ecology, Evolution, and Systematics, vol. 38, 2007, pp. 155–178. Illiad, doi:10.1146/annurev.ecolsys.38.091206.095.

[8] Schofield , Heather. “Communication Between Animals.” Schofield Investigations, KSC Open , 1 Mar. 2018. schofieldinvestigations.kscopen.org/schofield-courses-00345/kscanimbehav/communication-between-animals/.

[9] Verbeek, Peter, et al. “Differences in Aggression between Wild-Type and Domesticated Fighting Fish Are Context Dependent.” Animal Behaviour, vol. 73, no. 1, 2007, pp. 75–83., doi:10.1016/j.anbehav.2006.03.012.

Required Materials:

6x  Betta Splendens ( 5 male and 1 female) all mature adults

6x 1 gallon tanks- one for each fish, for housing

1x 5 gallon tank with a divider  for mirror observations

1x opaque cover for divider to prevent interactions prior to the start of each trial

Small mirror that can be attached to the side of the tank

1x Fish food

1x Fish net

1x bottle of  API  Splendid Betta Complete Water Conditioner from Petco 

1x tank pebbles 

1x box of plastic sandwich bags to use for fish tank acclimation/transfers

 

Further Question: Do closely related species exhibit the same aggressive behaviors based upon the sex of bystanders?

 

Please follow and like:
error