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


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. 



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

Table 2: Sample data collection table.



[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,

[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,

[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

[4] Mohrman, Eric. “What Does Conditioner Do for an Aquarium?” Pets, The Nest, 2007,

[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,

[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.

[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?


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