Behavioral Responses of Javanese Medaka (Oryzias Javanicus) Versus Zebrafish (Danio Rerio) in Open Field Test.


 Background: Locomotion is integral for animal survivability. However, the understandings of locomotor that lead to exhibition of multiple complex behaviors of fish models in response to an open field environment still remain unresolved. To determine whether two different fish models, Javanese medaka and zebrafish have similar baseline locomotor activity in open field paradigm, an open field test was used. Results: Results showed that Javanese medaka exhibit increased in exploratory activity with lower anxiety responses; exhibit a steady habituation response in OFT paradigm and vice versa in zebrafish. Medaka also took longer duration to establish home-base in comparison to the zebrafish. Although no other motor responses were observed, both fish species displayed strong preference of left eye used to assess the OFT tank. Conclusion: Medaka exhibits slower locomotors activity, lower anxiety responses and steadily maintains its locomotion once they reached habituation. In comparison, zebrafish demonstrated bolder behavioral phenotypes where they showed faster locomotors activity, higher anxiety responses with similar habituation response to the Javanese medaka. Thus, this present study revealed that two different teleost aquatic model organisms, Javanese medaka and zebrafish have different behavioral phenotypes in open field test.

adapted to suit the nature of sh to be in the water, including OFT (Matsunaga and Watanabe, 2010; Baker et al., 2018).
OFT usually provides an index of general behavior in particular exploratory activity, which is a crucial response to novelty (Lynn and Brown, 2009;Rosemberg et al., 2011). Like rodents, zebra sh exhibit thigmotaxis, as initial response to OFT and increased exploratory activity, which spikily decreases during the trial, re ecting habituation had achieved (Leussis and Bolivar, 2006;Kysil et al., 2017). Thigmotaxis is preference of animals towards the periphery of an open eld arena and avoiding center area which ideally used to evaluate anxiety status (Schnörr et al., 2012). Animals tend to establish a reference place (homebase) which they return regularly after exploring the environment and spent more time during trial (Rosemberg et al., 2011;Stewart et al., 2012). Home-base formation has been reported to re ect a behavioral state comparable to thigmotaxis (Stewart et al., 2012). Mounting evidence shows that . Well-developed sensory motor and cognitive systems, high sensitivity to environmental stimuli, and quanti able behavioral phenotype highlights their strengths as emerging models for neurobehavioral research (Signore et al., 2009;Cachat et al., 2011;Kalueff et al., 2014). Thus, this will help to improve our fundamental understanding about their neurobehavioral responses before further manipulation in more advanced studies. Therefore, the aim of the current study was to compare behavioral responses of Javanese medaka (Oryzias javanicus) in comparison to zebra sh (Danio rerio) upon introduction into OFT. Speci cally, this study assess locomotor, habituation, exploratory, thigmotaxis and other motor responses for each sh models in the rst six minutes in the OFT paradigm.

Animal husbandry
All experiments were performed in accordance with the rule of Institutional Animal Care and Use Committee of Universiti Putra Malaysia (UPM) (UPM/IACUC/AUP-R049/2019). Wild-type short n adult zebra sh (Danio rerio) of mixed sex were purchased from local petshop (AquaMart, Kajang) and retained at Laboratory of Natural Products, Institute of Bioscience, UPM. The sh were maintained in aquarium water (de-chlorinated, UV treated, aged tap water) at 28°C, fed with brine shrimp (Artemia salinaa) four times daily and kept in a standard 14/10 hour light/dark cycle (Koerber and Kalishman, 2009). Adult Javanese medaka (Oryzias javanicus) were collected from an estuary located in Sungai Pelek, Sepang, Selangor (2.6416° N, 101.7148° E). Then, the sh were acclimatized to a freshwater environment by gradual reduction of salinity from 18.50 to 0.00 ppt in the same laboratory within two weeks (Aziz et al., 2017). Both sh species (n=30; size 3-5 cm) were acclimatized for 10 days in the laboratory condition in 40L top-ltered tank (61 cm length X 31 cm width X 31 cm height). Then, behavioral evaluations in OFT for both types of sh were conducted in a separate behavior room. Following behavioral recording, the shes were euthanized using 500 mg/L Tricane (Sigma-Aldrich, St.Louis, MO) for approximately 10 mins until there were no operculum movements observed (Strykowski and Schech, 2015).

Open eld test (OFT) setup
Open-eld test behavioral recordings were performed between 8.00 a.m and 2 p.m in a behavioral recording room under standard illumination (~1000-1100 lux). 24 hour prior to the behavioral assessment, 10 shes for each sh species were transferred into holding tanks (30.0 cm length X 16.0 cm width X 18.0 cm height) and were kept in the behavioral recording room. Then, each sh was carefully netted into the open eld test (OFT) tank for behavioral recording to evaluate the locomotor activity, thigmotaxis, home-base formation and other motor responses. Fish handling and all behavioral recording procedures were conducted by a single observer blinded to any treatment of the sh.
The sh behaviors in the OFT tank were recorded in a customized closed framework as shown in the Figure 1A for 6 minutes (modi ed from Nema et al., 2016). The closed framework was used to eliminate the in uence of outside interferences like the presence of experimenter from affecting the behavior of the sh. The behaviors of the shes were recorded for six mins, using a 18-megapixels DSLR (1200D, Canon Inc., Japan) equipped with 55 mm kit lens (set to video setting, with manual focus) that was placed at the top center of the tank at the distance of 90 cm from the bottom of the tank. A white corrugated plastic sheet was used to surround a customized light box (± 1000 lux) (55 cm length X 55 cm width X 10 cm height), in order to ensure uniform light distribution. The OFT tank (37 cm length X 29 cm width X 18 cm height) lled with aquarium water was placed on the top of the light box. At the end of the behavioral recording, the sh was removed from OFT tank for euthanization. OFT tank was rinsed and lled with new aquarium water before subsequent recording. The behavioral recordings for both types of sh species (n=30) were conducted on different day within the same week.

Locomotor behavior
The behavioral analysis was conducted o ine in a computer using ZebraLab 4.2 software (ViewPoint Life Sciences, France) to track the locomotor activity of the sh for 30 frame per sec (Crispim Junior et al., 2012; Audira et al.,2019). The video-tracking data were then used to evaluate relevant measures of exploration and habituation across time such as total distance moved and speed travelled. The total distance moved and speeds travelled for six mins trial were representing the total locomotor activity. To identify the exhibition of exploratory and habituation response, both total distance moved and speed travelled were divided into three phases such as early, mid and nal with two mins intervals for each time interval. Other endpoints that were measured during the test including time spent per area (center/middle), number of entries to the center, inactive count at the center and inactive duration at the center.

Thigmotaxis
Thigmotaxis or wall hugging is the preference of animal to avoid center of arena and move in closer to the edge of an open eld arena which used as anxiety index (Schnörr et al., 2012). For thigmotaxis data acquisition, the recorded videos were analyzed using ZebraLab 4.2 software (ViewPoint Life Sciences, France) and the regions of interest were drawn using the software into three main regions: outer, middle and center ( Figure 3A). Thigmotaxis was presented as the percentage of the total distance moved at the edge divided by total distance moved multiplying by a factor of 100 (Schnörr et al., 2012). The data were analyzed for every 1, 2 and 3 mins in order to identify the optimal time interval to quantify the thigmotaxis behavior. The analysis was separated based on time interval to prevent the in uence of exploratory behavior exhibited by the sh in the early period of recording.

Home-base formation
The home-base is represented as the section where the animals repeatedly return after exploring the surrounding and spent the longest time in the open eld arena (Eilam and Golani, 1989;Rosemberg et al., 2011).The trace of sh swimming behavior was generated by video-tracking system to visualize sh locomotor behavior and home-base formation by using ImageJ (https://imagej.nih.gov/ij/) plugin, AnimalTracker API (http://animaltracker.elte.hu/ plugins). This tracker plugin provides the X,Ycoordinates of the sh in each frame by processing the recorded videos (Gulyás et al., 2016). Then, the videos were imported into ImageJ as virtual stacks and converted into 8 bit gray scale. The processing chains of the recorded videos using ImageJ software were based on the previous studies by Gulyás et al., 2016. The X,Y-coordinates were then exported into Microsoft Excel as a text le. The videos with tracking and representative tracking were saved as AVI and JPEG format using ImageJ respectively. The representative tracking with higher number of movement at the edge of the OFT arena were used to represent the home-base formation of two different sh species across time (Rosemberg et al., 2011).

Other motor responses
Other motor responses exhibited by both sh model were also observed in the OFT tank. These responses were manually scored and analyzed by trained observers including: i) percentage of behaviors occurrence towards the OFT walls (dashing, dashing along paradigm, head contact and no dashing), ii) percentage of time spent for body posture orientation towards the OFT wall (left, right and frontal-vertical) and iii) percentage of body movement (clockwise) ( Figure 4A) (Kohda et al., 2019). The sh was considered exhibits dashing along when the body postures exposing either right or left side of the head towards the OFT walls. Head contact was considered when the sh orient its body in frontal-vertical postures exposing the head vertically towards the OFT walls. Meanwhile, dashing and stopping was considered when the sh orient its body either right or left to the OFT wall and stay immobile for approximately less than 0.1 sec. No occurrence of dashing as described above was classi ed as no dashing behavior.
Fish body movement were then determined as clockwise movement if the turn angle is positive value ranging from -180° to 180° (Rosemberg et al., 2011). All the X and Y coordinates obtained through a freeware automated AnimalTracker plugin, ImageJ were then exported and further analyzed using Microsoft Excel based on Equation as suggested by previous study (Nema et al., 2016). The absolute turn angle represents the sum of all vectors movement created from one position to animal's centroid to the next. The turn angle of sh between frames calculated as theta (θ) degree from the slope using Equation.
The slope was determined as the ratio of ∆Y and ∆ X. ∆X and ∆Y are the differences between locations of X and Y in successive frames respectively. X next , X previous and Y next , Y previous are successive X and Y frames respectively.

Statistical analysis
The statistical analyses were conducted using SPSS (SPSS v.22.0). Two-way analysis of variance (ANOVA) was used to determine the differences of behavioral responses between two adult sh species, Javanese medaka and zebra sh in OFT. Dunnet test was set at p<0.05 as the threshold for statistical signi cance to compare behavioral responses between two different sh species and within different time intervals. Data were represented as average values ± standard error of mean (SEM). GraphPad Prism 6.0 version statistical software (GraphPad Software, USA) was used for all graphs. The parameters that were observed include exploratory and habituation response (total distance moved, speed travelled), anxiety-like behavior (percentage of thigmotaxis) and aggressive behaviors [i) percentage of occurrence of behaviors: dashing along the paradigm, head contact with paradigm, dashing and stopping and no dashing; ii) percentage of time spent for different body posture: left, right and frontal vertical, iii) percentage of clockwise body movement].

Spontaneous locomotor activity
Javanese medaka exhibited lower locomotor activity and vice versa for zebra sh over 6 mins behavioral recording using OFT tank. Figure 1B showed the representative swimming tracking of Javanese medaka and zebra sh during 6 mins that was divided into three time intervals.  Figure 1C-D). The total distance moved for Javanese medaka was three times lower than zebra sh while swimming speed was two-fold lower than zebra sh. Even though the total distance moved and swimming speed for Javanese medaka and zebra sh increased and decreased respectively over time, however, there were no overlapping observed throughout the recording time ( Figure 1E-F).
Spontaneous locomotor activity was analyzed based on 2 mins time interval where the rst 2 mins (early phase) was considered as exploratory phase and the remaining 4 mins (mid and nal phase) was considered as habituation responses. During the rst 2 mins in the OFT, Javanese medaka displayed an increment of spontaneous locomotor activity with time in contradictions to zebra sh ( Figure 1E-F). Javanese medaka demonstrated a gradual increased while zebra sh exhibited a moderate decreased in spontaneous locomotor activity. Both sh species reached a plateau in their locomotion at mid-phase, showing that both sh species started to habituate in the OFT ( Figure 1E-F). These ndings suggest that Javanese medaka and zebra sh have different baseline locomotor activity in a novel environment.

Thigmotaxis and home-base formation
Thigmotaxis is a tendency of an animal to move in contact with vertical surface and commonly moves towards the periphery of a novel arena avoiding center area. Thigmotaxis was measured as percentage of total distance moved in outer area over total time duration. To compare thigmotaxis in both Javanese medaka and zebra sh in different time frames (every 1-6 mins intervals), two way ANOVA and Dunn-Sidak test were performed. Results showed that thigmotaxis for every 1 mins intervals in Javanese medaka were signi cantly increased at 240 s (82.65 ± 5.99 %) until 360 s (88.55 ± 5.99 %) (Figure 2A). Meanwhile, thigmotaxis in zebra sh also increased signi cantly at later time intervals, 300 s (82.51± 5.89 %) until 360 s (80.71 ± 5.89 %). For every cumulative 2 mins intervals, Javanese medaka exhibited a gradual increased in thigmotaxis from 54.696 ± 5.315 % to 88.1796 ± 5.315 %. On the other hand, thigmotaxis percentage of zebra sh increased from 60.19 ± 5.225 % to 81.6066 ± 5.225 % ( Figure 2B). Data analysis using cumulative 3 mins intervals for both sh species tested, showed a prominent increment trend in thigmotaxis from 59.8709 ± 5.126 % to 86.3366 ± 5.126 % for Javanese medaka and 64.244 ± 5.083 % to 79.7248 ±5.083 % for zebra sh ( Figure 2C). Since both sh species started to display a signi cant edge preference after 3 mins, therefore, these ndings suggest that the most ideal approach to analyze thigmotaxis behavior in OFT could be after 3 mins behavioral recording. Generally, both sh species showed increased percentage of thigmotaxis behavior with time. Figure 2D-O showed representative swimming tracking in the OFT tank for every one min for both sh species. In the rst minute, Javanese medaka remained immobile for at least 23 sec at the edge, and then immediately swam to the center and middle region. After that, Javanese medaka swims towards the edge of the OFT swimming arena ( Figure 2D, additional le 1). In comparison to medaka, zebra sh immediately swam towards every edge of the OFT tank within the rst min ( Figure 2J, additional le 2). Formation of home-base by Javanese medaka (Figure 2F-I) and zebra sh ( Figure 2N-O) were observed when they notably showed a prominent preference towards the edge of similar location. A distinct formation of home-base started to form once they were habituated to the OFT tank. The home-base formation was represented by the highest tracking movement formed at the edge of the OFT tank. Figure 3B showed that Javanese medaka exhibited insigni cant number of entries to the center, while zebra sh showed a signi cant decreased. These results showed that both sh species still utilized the middle and center regions of the swimming arena as horizontal transition to explore the OFT tank. During the rst min, Javanese medaka remained inactive for more than 16.53 ± 1.956 sec at the center. In comparison to zebra sh, they spent less than 0.2433 ± 1.956 sec at the center before they immediately swam towards the edge of OFT tank ( Figure 3C). Meanwhile, in the rst 60 s in OFT, Javanese medaka displays a 30-fold higher number of inactive counts at center region (3.690 ± 0.6520) in comparison to zebra sh (0.1333 ± 0.6520) ( Figure 3D). In the subsequent time intervals, Javanese medaka display 2fold increment of inactive count at 120 s (6.172 ± 0.6520) and decreased over 6 mins time. In contrast, zebra sh displayed almost zero inactive count at the center throughout the 6 mins (0.0 -0.3333 ± 0.6520). These results suggest that Javanese medaka were less active, calmer and have lower anxiety responses. In comparison, zebra sh were found to be more active, aggressive and have higher anxiety responses.

Other motor responses
To evaluate other motor responses for both type of shes, different parameters were used including i) dashing along OFT wall, ii) head contact to the OFT wall, iii) dashing and stopping performed alongside the OFT wall and iv) other behaviors ( Figure 4A). Javanese medaka exhibited highest occurrence of head contact with the wall of the OFT (46.31 ± 3.832 %), followed by dashing along the wall (42.41 ± 3.832 %), dashing and stopping (5.103 ± 3.832 %) and other behaviors (6.138 ± 3.832 %) ( Figure 4B). Meanwhile, zebra sh immediately swam towards the edge of the OFT tank and exhibited dashing along the wall of OFT (43.53 ± 3.832 %), followed by head contact with the wall (38.63 ± 3.832 %), dashing and stopping (9.600 ± 3.832 %) and other behaviors (8.333± 3.832 %) ( Figure 4B). Both sh species displayed a balance body posture as they spent left body orientations (Javanese medaka: 46.00 ± 3.030 %; zebra sh: 38.66 ± 3.030 %) and right body orientations (Javanese medaka: 37.64 ± 3.030 %; zebra sh: 40.93± 3.030 %) equally, over 6 mins trial to navigate and explore the OFT tank ( Figure 5B). In addition, they also demonstrated a frontal-vertical body posture to the OFT walls where both sh species had no signi cant different (Javanese medaka: 16.29 ± 3.030 %; zebra sh: 20.45± 3.030 %). Furthermore, this study also recorded sh body movement (clockwise or counterclockwise) to determines which eye (left or right, respectively) was used to view the edge of the OFT wall. Javanese medaka displayed a signi cant clockwise body orientation at the early phase in comparison to zebra sh (Javanese medaka: 28 Figure 4D). No signi cant differences were observed for anticlockwise orientation in both sh species (Supplementary Figure 1). Thus, this nding may indicate that both species have strong preferences to use their left eyes in assessing a novel environment.

Discussion
This study demonstrates the comparison of locomotor behaviors between two different sh models, Javanese medaka and zebra sh for 6 min using OFT. This assay is crucial in determining whether both sh species have similar baseline activity in an open eld-based paradigm. Three different time frames were used to measure the net activity for both sh species. Early phase was analyzed as exploratory behavior while the remaining time, (mid and nal phase) were used to evaluate the habituation responses. Results showed that Javanese medaka steadily increased their locomotor activity with lower anxiety responses in a novel-based paradigm. Meanwhile, zebra sh displayed a decreased in locomotor activity during early phase with higher anxiety responses.
Exploration plays an important role in animal's natural behavior that is mainly used to investigate novel environments and to sustain their survivability (Baker et al., 2018). Interestingly, Javanese medaka took a longer duration before exploring the OFT tank. These ndings suggest that Javanese medaka were characterized as passive individuals since they displayed shorter swimming distance and lower exploratory activities. In comparison to Javanese medaka, zebra sh have bolder behavioral phenotypes in open eld environment since they rapidly swam throughout the novel arena, thus resulting in longer swimming distance. Boldness and shyness primarily denote the willingness among individual to take risk particularly in novel environment (Sih et al., 2004;Mustafa et al., 2019). The apparent disparity behavior between sh species may have arisen due to variation between domesticated (laboratory) zebra sh and wild Javanese medaka population. This ndings corroborate with previous studies where Hutter et al., (2011) found that wild-derived zebra sh were more shy and distressed in captivity, whereas the domesticated zebra sh were regularly bold and eagerly approach human and other stimuli (Wright et al., 2006;Moretz et al., 2007). Besides, the captive environment which was highly stable with the absent of predators likely results in rapid domestication that leads to increase in boldness behavior in all captive vertebrates (Huntingford, 2004 , 2019). Noteworthy, the difference in boldness and risk taking behavior of wildtype Javanese medaka versus domesticated zebra sh could also have been in uenced by their genetic variation (Wright et al., 2006;Norton et al., 2011). However, further study should be conducted in the future to evaluate the differences of exploratory behavior for domesticated Javanese medaka versus wild caught Javanese medaka. A constant in the locomotor activity over time also implicated that the exploratory activity started to diminish due to exhibition of habituation response (Rankin et al., 2009).
Habituation is de ned as a behavioral response decrement which results from repetitive stimulation without involving sensory adaptation/sensory fatigue or motor fatigue (Rankin et al., 2009;Thompson, 2018). Notably, Javanese medaka took slightly longer time to habituate in the OFT tank, while zebra sh habituated faster to the OFT environment. Different rate of habituation within populations are probably shaped by natural selection which in uenced by animal's adaptation towards its natural habitat (Bell and Peeke, 2012). A steady habituation in Javanese medaka might be due to their preferences for shallow periphery of the rivers where water current is slower and away from any point of disturbances (Yusof et al., 2013). In contrast, rapid habituation observed in zebra sh likely due to their natural habitat selection in the wild. They inhabit a wide range of habitats, including still, slow-owing and fast-owing water bodies (Suriyampola et al., 2016). Animals that experienced a more complex rearing environment during their development will habituate faster to novelty (Zimmermann et al., 2001). Moreover, this active learning process may help sh to distinguish false alarms to harmless events involving different mechanisms depending on stimulation, sensory pathway, and signal processing which eventually trigger various exploration responses in sh (Raderschall et al., 2011;Godwin et al., 2012;Gaspary et al., 2018). Variations in natural habitat preference and adaptation for different sh species reduce the risk of predation and increase their survivability. Natural habitat variations between two different sh species might in uenced their habituations response in the OFT tank.
Thigmotaxis is a preference of animals towards the periphery of a novel arena and avoiding center area which ideally used as anxiety index (Schnörr et al., 2012). In the present study, Javanese medaka were found to be passive and have lower anxiety responses in comparison to zebra sh that were active and more aggressive, with higher anxiety responses. A signi cant edge preference started to be displayed by both sh species after 3 min, suggesting that the ideal time to analyze thigmotaxis behavior in OFT should be measured starting from fourth mins in OFT. The early phase of behavioral recording in a novel environment is less suitable for thigmotaxis assessment due to the in uence of exploratory behavior (Gould et  clearly displayed thigmotaxic behavior. The preference to the periphery proposed that both sh species utilized vertical surfaces of the OFT wall as a spatial clue for navigation. This similarity of behavioral strategies for both sh species and rodents in novel environment suggest that OFT walls serve as a guiding and attractive force on locomotion which modulates their direction and speed in reference to walls during OFT navigation (Horev et al., 2007). These forms of attraction were suggested involving recognition and locational memory which may also underlie the Javanese medaka and zebra sh locomotion found in this study (Stewart et al., 2010). However, anxiety-related neuroendocrine responses should be taken into consideration in the future study by analyzing whole-body cortisol levels (Yeh et al., 2013). This will further con rm whether exposure to open eld environment altered anxiety responses in both sh species, physiologically.
Furthermore, this study discovered that the formation of the home-base for both sh species were established at the edge of the OFT. The nding revealed that both sh species have one prominent homebase formation that was commonly established close to the OFT walls. Worth mentioning, Javanese medaka takes slightly longer duration to establish a home-base in comparison to zebra sh. A distinct formation of home-base started to form once they were habituated to the OFT tank. The home-base is denoting as a place in the area for which the experimental animal shows a preference across time, With respect to other motor responses in OFT, both sh species displayed a balance left and right body orientations in order to navigate and explore OFT tank. No signi cant different in the percentage of frontal-vertical body posture towards the OFT walls were observed for both sh species. This frontalvertical body posture suggested that the sh exhibit a pause and look behavior. This behavior is an important aspect of exploration that allows information gathering which required for decision making during their exploration in novel environment (Kalueff et al., 2014;Redish, 2016). It is worth to note that the formation of sh re ection on the OFT wall made up of transparent acrylic sheet during the behavioral recording was observed. This study was accidentally found that zebra sh responded aggressively to its own re ection on the wall of OFT tank in comparison to Javanese medaka. Nonetheless, this study was unable to determine whether the sh can view its own re ection on the acrylic sheet. Therefore, to determine whether the sh have self-recognition, this visually mediated behaviors can be further explored for future research by tracking eye movement using optokinetic re ex (OKR), oculomotor behavior and a built in customized paradigm specialized for self-recognition assessment should be as well implement (Maurer, et al., 2011;Dehmelt et al., 2018).
This study also assessed sh body movement (clockwise or counterclockwise) to determine which eye (left or right, respectively) was used to view the edge of the OFT wall. Both Javanese medaka and zebra sh signi cantly displayed lower than 50% of clockwise body movement throughout early, mid and nal phase, suggesting that both sh species have strong preference to use their left eye. No signi cant differences were observed for anticlockwise orientation (right eye) in both sh species (additional le 3).
In accordance with the present study, previous studies have reported that adult zebra sh used their left eyes to assess the novelty of objects or environment whereas right eyes were used for prey detection (Miklósi, et al., 1997;Sovrano, 2004;Watkins et al., 2004;Sovrano and Andrew, 2006).
This study showed that 6 min duration for behavioral recording in OFT was su cient for both Javanese medaka and zebra sh to exhibit a visible thigmotaxis and home-bases behavior. A previous study showed that zebra sh exhibited a home-base formation after 30 min observation time (Stewart et al., 2011). However, longer OFT recording time may be applicable to enhance the characterization of locomotor that lead to multiple complex behaviors in both Javanese medaka and zebra sh that remain to be discovered. Although top view for video recording provides an accurate tracking and quanti cation for both Javanese medaka and zebra sh behavior, further studies may utilize multiple cameras to generate three-dimensional behavior tracking. Moreover, manipulation of neuropharmacological agents will helps to increase our knowledge about home-base and locomotor phenotypes in both sh species. A previous study has shown that rodents demonstrated alteration in home-base speci c locations when drug were given in different doses (Dvorkin et al., 2010). Therefore, the sensitivity of both sh species towards neuropharmacological treatment on their behavior in novel OFT merits further investigation.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Authors' contributions NSAMS and NAB together conceived the idea, performed the laboratory work, analyzed and interpreted the data, and wrote the manuscript. NFR and NASH conducted behavioral recording procedures as single observer blinded to any treatment of the sh. WNWI supervised the project. All the authors have read and approved the manuscript. travelled of Javanese medaka were slower compared to zebra sh over 6 minute trials. Decrement in locomotion represents reduction in exploration of a sh species. (E-F) Total distances and speed travelled in medaka were gradually increased during early phase and remain constants for the remaining of experimental period. In the other hand, total distances and speed travelled in zebra sh were decreased during remaining phase. Exploratory activity during early phase and habituation response of zebra sh in a novel arena during mid and nal phase was higher than Javanese medaka. *Signi cantly different from Javanese medaka (P ≤ 0.05, n=30 for each sh species).  Javanese medaka which exhibit a uctuation. These showed that both shes may use the center and middle region as horizontal transitions to explore the OFT tank. (C) In the rst 1 min, Javanese medaka remained inactive for more than 15 sec at the center showing that it steadily swum to the center in comparison to zebra sh which spent less than ~0.01 sec at the center and immediately swum toward the edge of OFT. (D) Medaka having higher inactive count in the center may represent they were less active or calmer than zebra sh. Since zebra sh achieved almost zero in inactive count at the centre and actively swimming to the other regions of OFT. *Signi cantly different from Javanese medaka (P ≤ 0.05, n=30 for each sh species). Other motor responses (A) Schematic diagrams for body postures orientation in OFT paradigm representing occurrence of other motor response: dashing along and head contact (B-C) No signi cant difference in occurrence of behaviors in a novel environment over 6 min and time body posture towards OFT wall between Javanese medaka and zebra sh. These showed that there were no signi cant difference of aggression between these two sh species (D) Both Javanese medaka and zebra sh displayed lower than 50% of clockwise body orientation throughout early, mid and nal phase indicating