Observations of Agonistic Behavior between Two Male Gobies Acentrogobius virgatulus (Jordan & Snyder, 1901), in Maizuru Bay, Kyoto Prefecture, Japan

. Acentrogobius virgatulus (Jordan & Snyder, 1901) is a small coastal species of goby found along the Western Pacifc. It is commonly found in Maizuru bay along the muddy sediment between the intertidal zone and depths of 10m. In June and July of 2022, two independent agonistic interactions between male A. virgatulus were observed and recorded during its spawning season. One interaction, lasting over 4 minutes, included certain aggressive behaviors such as jaw locking, mouth gaping, fn extensions, rapid color changes, and fast strikes to the head and body. Another interaction exhibited similar mouth gaping, fn extension, and rapid color changes but did not lead to further escalation. Tese behaviors coincide with those found in similar species and provides in situ evidence of these uncommon interactions. Tis is the frst record of agonistic behavior by an Acentrogobius species. Accumulating fndings such as these can contextualize intraspecifc interactions, reveal diferences across multiple species, and guide future experiments.


Introduction
Goby agonistic behavior between conspecifcs is substantially examined in both captive and in situ observations and behaviors vary greatly among diferent species [1,2]. Tere are many circumstances associated with fsh agonism [3]. A common association is with territorial or sexual competition during spawning seasons, which is particularly present in sand and mud dwelling gobies such as Amblyeleotris japonica, Gobiusculus favescens, Lepidogobuis lepidus, and Eucyclogobius newberryi [4][5][6][7]. In most cases, size is a deciding factor of success and deters most agonistic interactions between competitors [2,5,[7][8][9]. It is when conspecifcs are of similar size that aggressive interactions escalate and diferent mechanisms for competition can be observed and documented [7]. Tese kinds of interactions are rare and not easily replicated in tank environments [10]. While there are many commonalities in behaviors among families, ethograms of specifc species can be unique. Documenting more species behaviors and the environments they are observed in could underscore their diferences and similarities. In situ observations and experiments also avoid the unwanted habituation of focal fsh in captivity which may perverse natural behaviors [11]. Furthermore, in situ experiments are seen as having higher ecological validity and can provide more valuable insights, as they can be easily compared to previously documented research [4,9,12]. Here, we examine an interaction captured in the natural environment of a species of Acentrogobius goby that has not been documented in peer-reviewed literature.
Acentrogobius virgatulus [13] is a small goby reaching 8 cm total length [14]. It ranges from Hokkaido down to the southern Kyushu Islands of Japan, including the east coast of the Korean Peninsula and Eastern China Sea [14,15]. Tis species of Acentrogobius prefers high salinity, muddy to coarse sand environments, and inhabits the intertidal zone to depths of 10 m, spatially between its closely related species [15][16][17][18]; Acentrogobius sp. 2 [14] and Acentrogobius pfaumii [19]. According to a 2010 survey conducted in Maizuru Bay [20], the spawning season for A. virgatulus lasts about two and a half months, from early June to late August, while the water temperature is between 14.7 and 30.1°C. Acentrogobius virgatulus typically spawn under substrate such as brick or roofng tiles [21] and have a facilitative relationship with Alpheus shrimp burrows [16,18]. Tis study reports for the frst time the agonistic behavior of A. virgatulus observed during routine underwater visual censuses in Maizuru Bay, in the Sea of Japan.

Materials and Methods
Routine underwater visual censuses are conducted twice a month while diving on SCUBA along the coast of Maizuru Fisheries Research Station, Kyoto University, Maizuru Bay (35°29′21.5″N 135°22′02.7″E) between depths of 1-12 m [22]. Censuses follow the coastline and survey the various environments and structural (natural and artifcial) outcroppings in the area. Water visibility ranges from 1 to 10 meters. Te seabed consists of a shallow oyster shell shelf that quickly drops of into a muddy substrate at about 5 meters. Tis muddy substrate is typically bare but contains scatterings of oyster shell clusters, artifcial reefs, and other anthropogenic debris. Alpheus shrimp burrows are common along the substrate and are commonly cohabitated by Acentrogobius species and Cryptocentrus flifer [23]. Video cameras (Olympus Tough TG5; Japan) in underwater housings are used to document irregularities or unknown species for later identifcation. Videos are taken of any unusual behaviors on each dive in order to reference in later reports as a secondary objective. Oceanographic data is collected daily from the same location [24].

Results
Te frst author observed and recorded on video an agonistic interaction between two male A. virgatulus living in the silty substrate along the east side of Maizuru Fisheries Research Station (35°29′22.6″N 135°22′07.2″E). Two cases of agonistic interaction of A. virgatulus totaling 330 secs were observed from April to September 2022.
Te frst recording was taken around 11:00 on 23 June 2022 at a depth of 5 m, 21.5°C, and a salinity of 34.0 PSU. Several Acentrogobius spp. were observed while SCUBA diving along the benthos. In contrast, two larger males of approximately 7 cm each were engaged in a jaw lock upon recording (ESM Video S1; Figure 1(a)). Other, smaller A. virgatulus were in the area during the encounter. In this instance, the fsh on the viewer's left attempts to break the lock while the fsh on the viewer's right attempts to maintain the lock (Figure 1(b)). Tis is the only jaw locking behavior the fsh display during the encounter. After 75 secs, the lock is broken, and the fsh on the right that had attempted to maintain the lock fees to a patch of algae. Te fsh on the left attempts to chase the feeing fsh until it escapes momentarily (Figure 1(c)). After 90 secs, the escaped fsh returns, and the two begin a series of repetitious lateral displays, mouth gaping, encircling, fn extension (Figure 1(d)), and fnally strikes to the head and body using the individual's mouth (Figure 1(e)). It is through these quick rapid strikes that distinction between each fsh is deemed unfeasible. During this encounter, the two individuals undergo distinct color changes from their normal coloration, notably lighter body colors (Figures 2(a) and 2(b)). After 93 secs of agonistic behavior and 12 distinct strikes, one of the specimens swims away from the area and back into the patch of algae nearby, ending the interaction. Te total interaction, from the frst observation, lasted 258 seconds.
A second recording was taken at approximately 10:30 on 25 July 2022 at a depth of 5 m, 26.4°C, and salinity 33.3 PSU. In this second recording, similar lateral displays, mouth gaping, and rapid color changes lasting 72 sec were observed between two male A. virgatulus but did not further escalate to jaw locking or strikes to the head and body (Figure 1

Discussion
Each of the subject A. virgatulus observed was identifed as males through the distinct dark margins on the anal, pelvic, and lower caudal fns, which are colorless in females [25]. While this behavior has not previously been reported in this species, similarly related taxa in the family Gobiidae are known to exhibit territorial aggression in similar fashion. Many of these behaviors have been examined in both laboratory settings (lab-based) and feld observations (in situ). One example being Eviota storthynx (lab-based), exhibiting male on male aggression similar to the actions seen in A. virgatulus in the form of mouth gaping, lateral displays, fn extension, rapid color change, and intentional strikes to the head and body [26]. Tese behaviors are associated with many species of goby in regard to male-male competition during spawning seasons, as with Rhinogobiops nicholsii (in situ), Amblyeleotris japonica (in situ), Pomatoschistus minutus (lab-based), and Bathygobius soporator (lab-based), and it is noted that larger sized males typically see success in these interactions [7,8,27,28].
However, these behaviors are not uniform among all gobies. Behaviors such as butting and head-beating have been documented in Paragobiodon sp. (in situ) as well as gape pushing and parallel swimming in Microgobius gulosus (in situ) [29,30]. In Gymnogobius heptacanthus (in situ), Gobiusculus favescens (in situ) (lab-based), and Eucyclogobius newberryi (lab-based), the females sometimes maintain territory and exhibit agonistic behavior towards consexuals [4,6,9,31]. Tese studies show similarities in behavior types but exhibit unique diferences. Te diference between the two recordings of A. virgatulus can likely be attributed to the success and failure of deterrence signaling, which did or did not lead to the escalated aggression of striking and jaw locking. In many species, the encircling lateral display and mouth gaping allow them to primarily present size and deter aggression from smaller conspecifcs [32] as seen in lab-based [10] and in situ observations [7]. It should be acknowledged that the signaling behaviors of A. virgatulus are more conspicuous and not as easily identifed as jaw locking and striking during the SCUBA surveys.
Of these studies, the key diference in experimental testing comes down to laboratory-based and in situ observational experiments. Several species of sand dwelling gobies have been studied in both contexts. Wacker et al. [9] examined Gobiusculus favescens aggressive behaviors through laboratory experiments in the context of what was described by feld observational experiments conducted by Forsgren et al. [4], building on previous in situ experiments examining its agonistic behaviors in western Europe. Similarly, the aggressive behavior of the sand goby Pomatoschistus minutus has been extensively studied in aquaria which was aided by earlier agonistic behaviors examined in situ [8,33,34]. Swenson [6] primarily studied behaviors of Eucyclogobius newberryi in captive conditions and also made opportunistic observations of aggressive behaviors while in the feld, citing similar feld observations made by Swift et al. [35]. Field studies are likely limited to species that are more easily accessible and could be further limited by environmental conditions. However, it is argued as being the more ecologically valid method when there is a currently overwhelming reliance on lab-based research in fsh cognition [12]. While A. virgatulus belongs to the Gobiopsis lineage [36], no studies have documented the behavior or territoriality in the species belonging to this lineage. Tese present observations could also advise subsequent tank experiments for similar gobies in the western Pacifc.
Further research could identify the factors resulting in this agonistic behavior and the reasons for such aggression. Such events of agonism between conspecifcs are rare and not easily documented in marine environments due to the absence of draws such as baited underwater cameras [37] (in situ). It is argued that in situ studies are the best way to understand reproductive and social dynamics, albeit difcult to conduct in the marine environment and can be data limited [38] (in situ). Field observations such as this one give baselines for further experiments examining these behaviors. By further identifying factors and habitat association of these behaviors, future behavioral experiments can tailor data collection methods such as remote cameras to specifc environments. Observational feld studies can contextualize these social dynamics, highlight diferences across multiple species, and guide future experiments.

Data Availability
All the available data are provided within the supporting fgures and Electronic Supplementary Data. Oceanographic information can be provided upon request from the corresponding authors.

Conflicts of Interest
Te authors declare that there are no conficts of interest.