On the surface or down below: Field observations reveal a high degree of surface activity in a burrowing crayfish, the Little Brown Mudbug (Lacunicambarus thomai)

Kaine M Diehl; Nicoleena M Storer; Hogan D Wells; Destinee A Davis; Zachary J Loughman; Zackary A Graham

doi:10.1371/journal.pone.0273540

. 2022 Oct 14;17(10):e0273540. doi: 10.1371/journal.pone.0273540

On the surface or down below: Field observations reveal a high degree of surface activity in a burrowing crayfish, the Little Brown Mudbug (Lacunicambarus thomai)

Kaine M Diehl ¹, Nicoleena M Storer ¹, Hogan D Wells ¹, Destinee A Davis ¹, Zachary J Loughman ¹, Zackary A Graham ^1,^*

Editor: Louis-Felix Bersier²

PMCID: PMC9565396 PMID: 36240144

Abstract

Opposed to most crayfish species that inhabit permanent bodies of water, a unique burrowing lifestyle has evolved several times throughout the crayfish phylogeny. Burrowing crayfish are considered to be semi-terrestrial, as they burrow to the groundwater—creating complex burrows that occasionally reach 3 m in depth. Because burrowing crayfishes spend most of their lives within their burrow, we lack a basic understanding of the behavior and natural history of these species. However, recent work suggests that burrowing crayfishes may exhibit a higher level of surface activity than previously thought. In the current study, we conducted a behavioral study of the Little Brown Mudbug, Lacunicambarus thomai using video surveillance to determine their degree of surface activity and behavioral patterns. Throughout 664 hrs of footage, we observed a surprisingly high amount of activity at the surface of their burrows—both during the day and night. The percentage of time that individual crayfish was observed at the surface ranged from 21% to 69% per individual, with an average of 42.48% of the time spent at the surface across all crayfish. Additionally, we created an ethogram based on six observed behaviors and found that each behavior had a strong circadian effect. For example, we only observed a single observation of foraging on vegetation during the day, whereas 270 observations of this behavior were documented at night. Overall, our results suggest that burrowing crayfishes may exhibit higher levels of surface activity than previously thought. To increase our understanding of burrowing crayfish behaviors ecology, we encourage the continued use of video-recorded observations in the field and the laboratory.

Introduction

Crayfish are a diverse group of freshwater crustaceans with over 700 species globally [1, 2]. Many crayfishes are considered to be model organisms for behavioral biology due to their ease of capture, suitability for laboratory studies, simple neural architecture, and complex behavioral repertoires [3–7]. However, this work stems from studies of a few model species from North America (i.e., Procambarus clarkii, Procambarus virginalis, Faxonius virilis); all of which inhabit lentic and lotic aquatic environments. Although most crayfish species inhabit surface water systems like streams, lakes, rivers, ponds, and marshes, crayfishes have repeatedly evolved a semi-terrestrial burrowing lifestyle throughout their phylogeny [8–10]. Despite all species of crayfish possessing the ability to burrow to some degree [11], the complexity and reliance on these burrows vary greatly both within and between species [12, 13].

Crayfishes have historically been assigned to three different ecological classifications based on their burrowing ability and their reliance on their burrows: tertiary, secondary-, and primary burrowing species [12, 14]. Tertiary burrowing crayfishes are species which rely on permanent bodies of water and are only capable on constructing relatively simple burrows underneath benthic substrates. Secondary burrowing species can also inhabit permanent bodies of water, but during periods of drawdown they are capable of excavating complex burrows to access groundwater. Primary burrowing species (hereafter referred to as burrowing crayfish) are often considered semi-terrestrial as they are highly reliant on their burrows for their entire life; as they potentially serve as a location for breeding, refuge from predators, and as a location to acquire and cache food [12, 13]. Burrowing crayfish burrows are often extremely complex and may have several entrances (i.e., portals), tunnels, and chambers that can reach several meters deep [12, 13]. In addition to providing shelter for the crayfish that construct them, such burrows can serve as necessary habitat for other organisms [15–18] and increase soil bioturbation and habitat complexity [19–21].

Compared to our knowledge of tertiary and secondary crayfishes that occur in lentic and lotic environments, our understanding of burrowing crayfish behavior is scant [13, 22]. Historically, burrowing crayfish were thought to spend nearly their entire lives below the surface, and thought to occasionally leave their burrows for brief terrestrial excursions to search for food or to find a mate [12]. Minimal surface activity and their propensity to retreat to their burrow when startled has led to difficulties in capturing and observing burrowing species [23, 24]. Resultant of their reclusive lifestyle, our understanding of burrowing crayfish activity patterns and behavior is minimal, with much of the work on these species being derived from in situ natural history observations [12, 25–29] and not systematic investigations. However, despite these difficulties, semi-naturalistic mesocosms and custom-built observation chambers have been useful in the study of burrowing crayfish behavior [30–34].

Burrowing crayfish are considered to be nocturnal [35], which further complicates our ability to adequately study their behavior. But in some instances, burrowing crayfish are observed at the surface of their burrow during the daytime [12, 35–37]. Interestingly, recent studies reveal that burrowing crayfish may exhibit a higher level of surface activity than previously thought [36, 38]. Bearden et al. (2020) conducted the most comprehensive study to date on the activity of burrowing crayfish [38]. Using motion-trigger laser photography in two species of burrowing crayfish (Lacunicambarus erythrodactylus and Procambarus holifieldi), Bearden and colleagues measured activity throughout a one-year period. In their study, activity was considered based on whether the crayfish was visible at the entrance of their burrow [38]. This study confirmed that nocturnal activity occurred most frequently, specifically during times of cool groundwater temperature and warm air temperatures [38]. Furthermore, despite observing substantial activity at night for both species, there were many occurrences of daytime surface activity throughout their study.

Bearden et al. (2020) provides robust evidence regarding the environmental correlates of activity in these two species and serves as a solid foundation to build off for future studies on burrowing crayfish surface activity. However, the use of photography and not videography limited their insights into crayfish surface behaviors. As such, the photographs made defining behaviors difficult, and limited behavior determination to chimney construction and surface activity [38]. As stated previously, Bearden et al. (2020) goals were not specific to defining burrowing crayfish surface behaviors, but rather determining if surface activity was correlated to specific environmental correlates. Therefore, the behavioral complexity of burrowing crayfishes still remains unknown. Given their utilization of complex burrow systems and evidence of social behavior [13], burrowing crayfishes species may exhibit complex ritualistic behaviors like non-burrowing crayfishes [3, 13, 39]. However, no study to date has conducted long-term behavioral observations of a burrowing species in a completely natural environment.

Here, we conducted a behavioral study of a burrowing crayfish species through video-recorded field observations. Specifically, we monitored the surface of burrows inhabited by the Little Brown Mudbug, Lacunicambarus thomai (Fig 1). Lacunicambarus thomai is as a burrowing crayfish species with a high propensity to inhabit burrows in marshes, roadside ditches, and flooded fields [40]. Populations of L. thomai often live in localized colonies with conspecifics and inhabit burrows that are relatively simple but can nonetheless be up to 1–1.5 m deep [40, 41]. Compared to most burrowing crayfish species, L. thomai and other members of the genus Lacunicambarus seem to exhibit a high degree of surface activity [36, 37, 40–44], which makes them ideal for our investigations. Overall, we hope to shed light on the surface behavior of L. thomai by 1) determining the prevalence of surface activity and 2) creating an ethogram specific to burrowing crayfish, and 3) analyzing the behavioral repertoire and prevalence of behaviors exhibited by L. thomai.

Fig 1 — The burrow’s entrance portal is out of sight due to the extensive leaf litter coverage. Photographs courtesy of John Freudenstein.

Methods

Study site

In June and July 2020, we conducted non-stop 24-hour video surveys within a population of the Little Brown Mudbug, Lacunicambarus thomai (Fig 1) in Hickory, Pennsylvania, U.S.A. This population of L. thomai is located on the edge of a man-made pond on a private, residential yard. More specifically, our study site was located in a dense patch of grasses on the edge of the pond with no canopy cover. Within this location, the water table remains shallow throughout the year and there is an estimated population of 20 L. thomai within an area of ~20 m². Burrows were sporadically located throughout the population and were ~1 m to ~ 5 m from one another. No other crayfish species have been collected or identified from this location and no crayfish have ever been collected from the pond. We chose which burrows to record based on evidence of recent surface activity (activity observed at burrow portal during preliminary night-time observation), as well as whether the burrow portal was visible from an overhead view. Because capturing burrowing crayfish often destroys the burrow and the population was relatively small, we chose to not capture the individuals. Therefore, we do not report morphological or demographic data from the recorded footage (i.e., sex, body size). However, based on the circumference of the burrow tunnels as well as the video footage, all burrows in which we recorded (see below) were from adult L. thomai. Importantly, although other burrowing crayfish species are known to exhibit social behavior and have multiple individuals within a single burrow, Lacunicambarus spp. seldomly exhibit social behavior and therefore it is assumed that each burrow only housed a single crayfish [41, but see 42].

Because our work was conducted on privately owned property on an unlisted crayfish species and we did not collect any specimens for this project, no specific permits were necessary for our research.

Videography

Overtop each burrow that we recorded, a custom-built video camera mount was constructed by placing a Reolink 5MP video camera inside of a 5-gallon bucket that we mounted with wire approximately 0.5 m above the burrow (Fig 2). These camera mounts protect the camera from the external environment and reduce glare. We recorded each crayfish until their burrow was either capped or until there was a video camera malfunction. If a crayfish burrow contained more than a one portal, we only mounted the video camera overtop the larger, primary portal which is where burrowing crayfish exhibit nighttime activity (Z. Loughman, personal communication). In total, we recorded and reviewed (see below) 664.7 hr of footage from six adult L. thomai (Table 1). The length of time recorded for each crayfish ranged from 44.5 6 hr to 263.82 hr (Table 1).

Fig 2 — (A) Diagram of our video surveillance setup used to monitor crayfish behavior. (B) Photograph of the setup *in situ* C) Photograph of the view of the camera mounted from inside of the bucket.

Table 1. Dates and total time that each crayfish was filmed throughout the duration of our study.

Only a single crayfish (Crayfish 5) was filmed discontinuously due to failure of the video recorder.

Crayfish
	Dates Filmed (MM/DD/YYYY)	Total Time Filmed (hr)
1	06/03/2020–07/02/2020	44.56
2	07/02/2020–7/20/2020	263.82
3	06/28/2020–06/30/2020	40.34
4	07/01/2020–07/06/2020	105.93
5	06/13/2020–06/14/2020; 06/28/2020–07/03/20	151.73
6	07/21/2020–07/24/2020	57.35
Combined	-	664.73

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Ethogram

We reviewed all video footage with Windows Movie Editor Software. While reviewing the footage for each crayfish, we created an ethogram based on observed behaviors (S1–S3 Files). In total, our ethogram contained 5 surface activity behaviors, as well as a single “inactive” behavior (under) which was used to denote when the crayfish was not visible at the surface of the burrow. See Table 2 for detailed descriptions of each behavior. Then, using our ethogram, we recorded what behavior each crayfish was exhibiting, the duration of the behavior, and the time in which the crayfish started and ended the behavior. We also noted whether each crayfish exhibited behaviors during the day or night based on the civil twilight time listed for Hickory, Pennsylvania, U.S.A (collected from www.time.unitarium.com). For our study, we considered a crayfish to be active if they were visible at the surface of their burrow (Fig 3; [35]). We considered a crayfish to be considered inactive if they were not visible at the surface of their burrow. We are unable to report on any below-surface activity within our study.

Table 2. Ethogram used to quantify the behaviors from recordings of Lacunicambarus thomai.

Behavior	Description
Rest–Claws Open	Crayfish is motionless and partially out of burrow portal with both claws in a “U” orientation directed anteriorly. Movements consist of simple shifts in body position.
Rest = Claws Joined	Crayfish is motionless and resting on burrow portal lip with both claws in a “V” orientation. Both claw tips touching or nearly touching directly anterior to the rostrum.
Forage	Crayfish is actively manipulating and foraging for plant material (leaves, grass, or roots).
Excavate	Crayfish is actively carrying, moving, or placing burrow substrate outside of the burrow beyond the burrow portal.
Hunt	Crayfish quickly rushes beyond the burrow portal in attempt to predate on living animal prey.
Under	The crayfish is inactive on the surface and not visible.

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Fig 3 — The top two images (A) and (B) represent crayfish in the rest–claws open posture, whereas the bottom two image (C) and (D) represent the crayfish in the rest–claws joined posture. Red circles and red lines are presented to give a representation of the differences in articulation between the claws open and the claws joined postures. Photos (A) and (C) were taken at night, whereas photos (B) and (D) were taken during the day.

Environmental data

Because we wanted to investigate broad-scale trends of how the surface activity of L. thomai varied with environmental conditions, we gathered environmental data from an online source (www.weatherunderground.com). We collected data from the closest available weather station in Imperial, Pennsylvania, U.S.A (~24 km away). Specifically, the environmental data that we collected from this source was air pressure, precipitation, temperature, and humidity. Later, these variables were used as covariates to determine which environmental variable best predicted L. thomai surface activity.

Statistical analysis

We conducted all statistical analyses in R version 3.5.1 [45]. For all of the models described below, we visually inspected our data using the performance package in R to assess normality and collinearity of our model fits [46]. For descriptive analyses of L. thomai behavior, we calculated the frequency, mean, and range of each behavior. Additionally, we wanted to describe the activity of L. thomai based on the time of day. Because of the uneven distribution of video footage length for each crayfish (Table 1), we decided to report the majority of our results in terms of percentages, and not raw values. Therefore, we calculated the percentage of crayfish which exhibited surface activity at each hour by dividing the number of hourly observations in which surface activity was observed by the total number of hours which we recorded throughout our entire study. We averaged these values together for all crayfish which enabled us to investigate patterns in surface activity based on the hour of the day. The same procedure was conducted to determine the percentage of time that each crayfish was observed at the surface throughout the daytime and nighttime.

To determine which environmental variables best related to burrowing crayfish activity, we initially used a model selection procedure with several linear mixed effect models with activity as dependent variable and the environmental variable (i.e., air pressure, precipitation, temperature, and humidity) as the independent variable. Visualizations of the environmental variables are depicted in the Supplemental Information (S1–S4 Figs). For this analysis, we determined a crayfish to be active during a given hour if they were recorded at the surface of their burrow within any point during that hour. In these models, we included crayfish ID as a random effect. Based on the Akaike information criterion (AICc) of these models [47], we found that only temperature and humidity effected activity of burrowing crayfish, and therefore we proceeded with these two variables for the remainder of this analysis.

Thus, to evaluate how environmental variables (i.e., humidity and temperature) and time of day (i.e., hour) influenced activity of L. thomai, we used multimodal averaging to determine how temperature, humidity, and time (independent variables) influenced the activity of crayfish (dependent variable). For this analysis, we determined a crayfish to be active during a given hour if they were recorded at the burrow portal within any point during the hour. Additionally, because there were multiple observations from each crayfish, we included crayfish ID as a random effect to control for individual differences in individual activity.

We first fit the full model to the data with the lme4 library of the R statistical package [48]. The full model contained temperature, humidity, time, and the interaction between these three variables as the independent variable. Because our dependent variable (i.e., activity) was coded as 1 (active) or 0 (inactive) we used a binomial distribution of error in this model. This model also included crayfish ID as a random variable. Then, we fit models that contained a subset of the terms in the full model. For each model, we calculated the corrected Akaike information criterion and the Akaike weight (i.e., the probability that the model described the data better than the other models in the set). Finally, we used the parameters of each model and Akaike weight to calculate a weighted average of each parameter. These model-averaged parameters enable one to determine the expected probability for any combination of values for the fixed factors in the full model. This approach eliminates the use of P values because all models (including the null model) contributed to the expected probabilities. Importantly, collinearity was unlikely to bias our model averaged parameters [49]; because none of our independent variables (humidity, temperature, and time) were not strongly correlated (all r² < 0.6).

To determine whether each specific behavior was more or less likely to occur during the day or night, we conducted six separate Chi-square tests of independence. We conducted 5 separate chi-squared tests for each individual behavior (relax, guard, forage, excavate, hunt, and under). We also performed a single chi-squared test for all six behaviors combined to determine whether all activity was more likely to occur during the day or the night. Additionally, we analyzed the likelihood of each behavior occurring during the day or night with a contingency mosaic plot [50]. Continency mosaic plots display the Pearson residual values as colors in relation to the deviation between expected (equally as likely to occur during the day and night) versus the observed frequencies [50]. This allows us to both visually and statistically investigate the directionality of the observed frequencies of each behavior (and the combined behaviors) in relation to equal frequencies.

Results

Hourly surface activity

We observed L. thomai exhibiting surface activity throughout every single hour of the day. However, there was variation in the times that L. thomai were most likely to be observed active at burrow portals. Surface activity peaked from 20:00 hr– 03:00 hr, with nearly every single crayfish active during those times during the study (Fig 4). After 03:00 hr, crayfish activity slowly declines with surface activity rarely being observed at 17:00hr. Despite being less active throughout the day, 25% to 50% of time each crayfish remained active throughout the daytime.

Fig 4 — Gray areas represent the civil twilight time for our study period that represents the transition between day and night. Crayfish were more likely to be active during the night than during the day. Data is reported from all crayfish throughout the entire period of our study.

Environmental predictors of surface activity

Lacunicambarus thomai surface activity was best predicted by a model containing the effect of time, humidity, temperature, an interaction between time and humidity, and an interaction between humidity and temperature (S1 Table). However, the three most likely models were all within 2 AICc values of the best fit model (S1 Table). Based on the model averaged coefficients, humidity, temperature, and the interaction between humidity and temperature all predicted the activity of L. thomai throughout the duration of our study (S2 Table). However, the humidity variable had the strongest overall effect (S2 Table).

Surface activity

Among the six crayfish observed, there was a high degree of surface level activity. Each crayfish spent at least 20% of the time that we filmed at their burrow portal. The percentage of total time each crayfish was observed at the surface ranged from 21% (9.18 hr) of the total filmed time to 69% (39.42 hr); with an average of 42% of the time spent active across all six crayfish (Fig 5). Interestingly, one of our six crayfish (crayfish 6) spent a greater portion of the time active above ground than underground. The remaining 5 L. thomai spent a greater percentage of time underground, with a range of 31% (17.92 hr) to 79% (120.47 hr) of their time spent underground and with an average of 58% of each crayfish’s time spent underground (Fig 5).

Fig 5 — We report the percentage of time that each crayfish individual spent active or inactive as well as a combined total for all six crayfish. For each crayfish, the total number of time (hours) that each crayfish was filmed is listed above each bar.

Day vs. night surface activity

All L. thomai exhibited a high degree of surface activity both during the day and during the night. The percentage of time that crayfish were active during the daytime ranged from 20% (7.71 hr) to 73% (13.65 hr) of the time with an average of 36.46% of activity occurring at the burrow portal or on the surface (Fig 6). Regarding nighttime activity, five of the six crayfish exhibited more nighttime activity at the surface of their burrow than they did daytime activity. Night activity ranged from 27% (5.15 hr) to 80% (30.46 hr) with an average of 63% of the total surface activity occurring during the night (Fig 6).

Fig 6 — We report the percentage of time that each individual crayfish spent during the day and the night as a combined total for all six crayfish. For each crayfish, the total number of time (hours) that each crayfish was filmed active is listed above the bar.

The behaviors exhibited by L. thomai varied depending on whether they were active during the day or night (S3 Table). The most common diurnal behavior recorded was the guard and relax behavior, which comprised nearly 80% of the total time crayfish were observed at the surface during the day (Fig 7). Interestingly, a single crayfish (crayfish 4) exhibited foraging behavior during the day for 0.001% (392 s) of the time that they were filmed. Because this was such a small portion, it is not visible in Fig 7A. We observed two crayfish (crayfish 1 and 5) excavating their burrow during the day. Overall, each crayfish spent most of their day activity exhibiting the guard behavior at their burrow portal (Fig 3; S3 Table).

Fig 7 — The percentage of time that each *Lacunicambarus thomai* spent exhibiting different behaviors throughout the (A) day and the (B) night. We report the percentage of time that each individual crayfish spent during the day and the night and combined six crayfish. The hunt behavior was not included in because of the relatively small time that this behavior was exhibited. For each crayfish, the total number of time (hours) that each crayfish was filmed active is listed above the bar.

Crayfish showed a greater level of nocturnal surface activity. The majority of their time was spent in the relax position during the night, and relaxing was more likely to occur during the night compared to during the day (Fig 7); both of these behaviors occurred at burrow portals. Interestingly, each crayfish spent a portion of their nighttime activity exhibiting foraging behavior, with an average of 11% of all night activity including foraging (Fig 7). The same two crayfish that excavated during the day (crayfish 1 and crayfish 5) were also observed excavating their burrow at night, but they excavated for a greater percentage of their nighttime activity than their daytime activity (Fig 7).

When analyzing the likelihood of each behavior occurring at day versus night, the chi-squared analyses demonstrated that every behavior was more likely to occur during one light period compared to another. Specifically, the guard behavior and inactivity (i.e., under) were more likely to occur during the day compared to the night (Fig 8; S4 Table). By contrast, excavate, forage, and the relax behavior were more likely to occur at night (Fig 8; S4 Table). The hunt behavior was also more likely to occur during the night than during the day (Fig 8; S4 Table).

Fig 8 — The width of the bars represents the number of observations that we recorded, whereas the height of the bars represents the proportion of observations that were recorded in the night (the bottom bar) versus the day (the top bar). The color of each bar represents whether the observed frequency of each behavior deviates from the expected frequencies if the variables were independent. Red denotes observed frequencies that are smaller than the expected frequency, whereas blue represents the observed frequencies that are larger than the expected frequencies. Gray represents a similar observed frequency when compared to the expected frequency.

Discussion

Out of the 664.73 hrs that we observed across six L. thomai; crayfish were active at the surface of their burrow nearly half of this time. Furthermore, we observed a surprisingly high amount of surface activity during the day, with nearly one-third of all surface activity occurring during the day and not at night. With our footage, we were also able to create an ethogram based on the five different surface behaviors (relaxed, guard, forage, excavate, and hunt) that we observed. Our ethogram allowed us to examine the behavioral repertoire and prevalence of the whether the observed behaviors more frequently occurred during the day or night. We discuss these findings in relation to previously published observations of burrowing crayfish surface behavior as well as potential opportunities for future research.

Before our study, little has been reported on the degree of surface activity and specific behaviors exhibited by burrowing crayfishes. Although surface activity has been observed throughout burrowing crayfishes both during the day and during the night, the prevalence in which L. thomai was active at the surface far exceeds that of other previously published reports. Previous accounts suggest that burrowing crayfish were only active above ground during a few instances which included high water events [51], during cloudy days according to light levels [36], or when looking for food or mates [12]. However, many of these accounts come from in situ natural history observations, and not systematic investigations. For example, Loughman et al. 2015 reports over 50 observations of Cambarus pauleyi at the surface of their burrow, but only three of these observations were made during the day [52]. Moreover, Foltz et al. 2018 states that Cambarus loughmani did not rise from burrows until a few hours after twilight, with no daylight activity reported [26].

Although we studied a small number of L. thomai during a two-month summer period, every crayfish was active during peak activity times. Only two previous studies have reported hourly activity data for burrowing crayfishes [35, 38]. For over a year, Bearden et al. (2021) collected hourly activity data for two burrowing crayfish species (Lacunicambarus erythrodactylus and Procambarus holifieldi). In their study, both L. erythrodactylus and P. holifieldi were observed active roughly 25% of the time during the night and were rarely observed being active during daytime hours [38]. In a laboratory study, Palaoro et al. 2013 collected hourly data on a South American burrowing species (Parastacus brasiliensis) and showed that this species spent a majority of their experiment within their burrow, but were more likely to leave their burrow during the night compared to during the day [35].

In our study, the high degree of surface activity observed may be specific to crayfish in the genus Lacunicambarus. However, because our study was conducted on few individuals, future research on the surface behavior of Lacunicambarus thomai and the other 13 species in the genus Lacunicambarus is warranted. Alternatively, other burrowing species (and genera) may also be highly active during the day, but researchers often exclusively collect burrowing crayfish during the night which can bias the prevalence of these behavioral observations [24, 51]. For example, in a seven year-long ecological study of the Digger Crayfish, Creaserinus fodiens, Norrocky et al. 1991 reported that all crayfish collections occurred at night [51]. Clearly, the high degree of surface activity that we found in L. thomai contrasts our previous understanding of the activity and surface behaviors of burrowing crayfishes. Further, our study solely reports observations of surface behavior, and we are unable to report on the presence, absence, or degree of underground activity.

Based on our analysis of the environmental predictors of surface activity, we found that humidity had the strongest overall effect of predicting burrowing crayfish surface activity. Specifically, we found that crayfish were more likely to be active at lower humidity values. These results contrast previous findings by Bearden et al. 2021, in which activity was best predicted by a combination of variables, including daylength, air temperature, and groundwater temperature. Importantly, our analysis only uses reported data from the macro-environment at a nearby weather station, and not local, micro-environmental variables (variables recorded at the exact environment of or study site), as was done by Bearden and colleagues [38]. For example, in their study, Bearden et al. (2021) found that chimney constructed was related to precipitation events. It remains unknown whether the other surface behaviors which we describe have similar correlations to environmental variables such as prescription or groundwater temperature. Future studies should take into account the variation within environmental variables such as air temperature and burrow temperature to investigate behavioral patterns in burrowing crayfish. For example, activity in burrowing mammals is often best predicted by an interaction between both surface and groundwater temperatures [53]. Therefore, similar surface and groundwater temperature interactions may occur in burrowing crayfishes which are already known to use their burrows as refuges from harsh environmental temperatures [12, 13, 38].

Besides our study, only one other study has created an ethogram specific to burrowing crayfish surface behavior, although this study was conducted in a laboratory environment [35]. By contrast, our in-situ recordings allowed us to create an ethogram based entirely on natural behaviors, which revealed the richness of burrowing crayfish surface behavior—many of which have been previously undocumented. In our study, because L. thomai were constantly being monitored, we were able to categorize each crayfish into one of five different surface behaviors. The two most prevalent activities were the relax behavior and the guard behavior, which we separated based on the orientation of the crayfish’s claws relative to their body position (Fig 3). Whether the guard behavior’s function is truly to defend the burrow is unknown, because we did not observe any intraspecific interactions in our study. However, defense, guarding-like behavior has been reported in other burrowing crayfish [51].

Interestingly, there were strong differences in the prevalence of the guard and relax behaviors depending on whether it was during the day or night. We found that during the day, L. thomai was more likely to be active in the guarded position compared to the relax position. By contrast, when we observed L. thomai active during the night, they were much more likely to be in the relax position than the guard position. The function of these different postural behaviors is unknown and requires further investigation. But we can speculate that the guard behavior is more likely to be exhibited during the day due to the increased potential for predator interactions. Indeed, we observed terrestrial predators (i.e., snakes and birds) interacting with L. thomai burrows during the day [37]. Alternatively, the rest behavior being more likely to occur during the night may relate to this posture’s increased ability to quickly capture live prey that move near the burrow [36, 37, 42]. Future studies must investigate whether or not these postural differences are present throughout other burrowing crayfishes, or if they are specific to L. thomai.

We also observed 271 instances of L. thomai engaging in active foraging for nearby vegetation. Within our observations, L. thomai often explored the area within the vicinity of their burrow portal and used their claws to cut nearby vegetation. Across all our observations, only a single instance of vegetation foraging occurred during the daytime, which demonstrates a strong preference for this behavior occurring at night when there are presumably fewer active predators. Interestingly, the vegetation was either immediately consumed at the base of the burrow or was taken down into the burrow out of the view of the camera. Therefore, our observations confirmed previous reports of burrowing crayfish foraging; because the area surrounding the burrow portals are often replete with vegetation [26]. Furthermore, crayfish burrows are commonly excavated into large chambers, and large amounts of vegetation are found within the burrow tunnels and the burrow chambers, which are likely to serve as a food storage cache [26, 27, 54]. Although our observations confirm that L. thomai does take vegetation down into the burrow it is difficult to confirm the hypothesis that L. thomai burrows may serve as a food cache. The use of crayfish burrowing chambers in laboratory settings [30] is required to confirm the possibility for crayfish using their burrows to store and consume food resources.

In addition to observations of L. thomai foraging for vegetation, we observed 32 instances of the crayfish exhibiting sit and wait hunting behavior. In these observations, the crayfish sat at the burrow portal and quickly lunged at live animal prey (i.e., the hunt behavior). In some instances, this ambush behavior successfully resulted in the capture of live animal prey, whereas other attempts did not result in successful acquisition of the prey. When this behavior resulted in a successful attempt, the crayfish immediately took the item into their burrow. This ambush behavior was more likely to occur nocturnally than diurnally, with only three of 32 observations occurring during the day. Interestingly, this sit and wait predatory behavior seems to be prevalent throughout the genus Lacunicambarus, as it has been reported in at least 2 other Lacunicambarus species [36, 37, 42]. Recently, Thoma (2022) reports that in Ohio, Lacunicambarus chimera is never active at the surface of their borrow, whereas another species, Lacunicambarus nebrascensis rarely exhibits surface activity [55]. Although other species of crayfish have been observed capturing live animal prey [28, 29], the degree and reliance on this ambush predation behavior across burrowing crayfishes is unknown.

Across the six crayfish that we studied, we only observed two L. thomai actively excavating their burrows. These observations indicate burrow excavation is not an exceedingly common L. thomai behavior [35, 38], and may only occur when necessary alterations to the burrow architecture are needed. Each observation of excavation behavior always occurred in a repeated series of ritualized behaviors. First, crayfish would be observed bringing mud up to the burrow portal. Once at the surface, the crayfish would immediately stop before reaching ground level and immediately tap their antennae at their edge of their burrow portal. Lastly, the crayfish would finally emerge and place the mud pellet and methodically use its claws the push the mud into position.

In previous studies of P. holifieldi, precipitation was one of the variables that influenced the likelihood of burrow excavation (i.e., referred to as chimney construction in the study) [38]. Indeed, burrows become flooded during times of rainfall and often require renovations following these events (Z. Loughman, personal observations). Only two small rain events occurred during our study period, which may explain the lack of burrow excavation being observed. Furthermore, excavation behavior was more likely to occur at night than during the day. Intuitively, burrow excavation is a relatively costly behavior, given substrates must repeatedly be excavated from the bottom of the burrow to the surface. Such behaviors are more likely to have increased costs during the day, both based on the high surface temperatures and the potential for increased rates of predation during the daytime.

In summary, based on our preliminary investigation, we found that L. thomai exhibits a high degree of surface activity. Further, we found that L. thomai exhibits several behaviors at the their burrow portal, and these behaviors all were more likely to occur during either day or night. However, our study brings several unanswered questions to light regarding the behavior of burrowing crayfishes. For example, although we observed many small foraging excursions that occurred within the immediate vicinity of the burrow, we did not observe a single large-scale excursion during this time. In other species of burrowing crayfishes, substantial surface activity occurs immediately after rainfall, and such crayfish may travel large distances in a short period of time [12; Z. Loughman & Z. Graham personal observations]. Although the exact function of these excursions is unknown, it is unclear whether or not L. thomai engages in such behaviors.

Further, throughout our study, we did not observe any intra-specific interactions. Again, scattered observations highlight the social behavior of burrowing crayfishes, but interestingly, we did not observe any social behavior throughout the course of our study. Future studies should conduct similar behavioral observations throughout the year and also investigate potential demographic differences in the behaviors we report. By expanding the time frame in which records occur, social interactions of larger foraging excursions may be recorded. Lastly, although our study has shed light on the activity of crayfishes at the surface, much remains unknown about the subsurface behavior of burrowing crayfishes. Additionally, since we discovered that this species displayed easily identifiable postural behaviors, this may allow for automated video analysis of body position, activity, and behaviors. As such, the continued use of burrowing crayfish observations chambers [30] will be paramount in furthering our understanding of the behavioral ecology of burrowing crayfishes. In conclusion, our study revealed a surprisingly high degree of surface activity in a species of burrowing crayfish. Based on the historical difficulties in studying burrowing crayfish, there is much to learn about the ecological and evolutionary dynamics of burrowing crayfish behavior.

Supporting information

S1 File. Recording of an excavation observation during the night.

(MP4)

Click here for additional data file.^{(15.8MB, mp4)}

S2 File. Recording of a foraging observation during the night.

(MP4)

Click here for additional data file.^{(87.5MB, mp4)}

S3 File. Recording of a hunt observation during the night.

(MP4)

Click here for additional data file.^{(45.4MB, mp4)}

S1 Table. The most likely model predicting the activity of burrowing crayfish based on time, humidity, temperature, an interaction between time and humidity, and an interaction between humidity and temperature.

(DOCX)

Click here for additional data file.^{(14.2KB, docx)}

S2 Table. Coefficients and standard errors (SE) for the model predicting the activity of L. thomai based on full model averaging.

(DOCX)

Click here for additional data file.^{(13.4KB, docx)}

S3 Table. The number of observations and the mean duration of each behavior observed during our study.

(DOCX)

Click here for additional data file.^{(13.7KB, docx)}

S4 Table. Results from Chi-squared test for the number of behaviors that occur during the day and versus the night.

(DOCX)

Click here for additional data file.^{(13.4KB, docx)}

S1 Fig. Average, minimum, and maximum air temperature at our study location in Hickory, PA throughout our study period (June 2020 –July 2020).

(PNG)

Click here for additional data file.^{(683.2KB, png)}

S2 Fig. Average, minimum, and maximum humidity at our study location in Hickory, PA throughout our study period (June 2020 –July 2020).

(PNG)

Click here for additional data file.^{(682.3KB, png)}

S3 Fig. Average, minimum, and maximum air pressure at our study location in Hickory, PA throughout our study period (June 2020 –July 2020).

(PNG)

Click here for additional data file.^{(577.4KB, png)}

S4 Fig. Precipitation at our study location in Hickory, PA throughout our study period (June 2020 –July 2020).

(PNG)

Click here for additional data file.^{(283.9KB, png)}

Acknowledgments

We thank the Davis family for the use of their land for the study location.

Data Availability

The data are available at Dryad at the following DOI: https://doi.org/10.5061/dryad.kh189328r.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0273540.r001

Decision Letter 0

Junhu Dai

3 May 2022

PONE-D-21-39709On the surface or down below: Field observations reveal a high degree of surface activity in a burrowing crayfish, the Little Brown Mudbug (Lacunicambarus thomai)PLOS ONE

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Reviewer #1: The authors made an interesting work, continuously filming the surface behavior of crayfish which were thought before to stay mostly or totally in underground burrows. They registered activity both during the day and night along with daily environmental factor variations and analyzed their behavior through the video recordings. While most behavioral studies in crayfish have been done in laboratory settings, this one contributes by rescuing ecological context and opening new perspectives of research. It is mostly well done but there are three general issues to be considered:

1) One of the big contributions of this work is to enable discrimination of individual behavior, which is not a simple task in natural observations. Individual data presentations are fine but I suspect that when average analysis are presented, some or most of them are not considering that one individual (#2) with more than double the number of data collection is biasing the results. This can be deduced from some graphs while others do not let us know because description of average calculations are missing in Materials and Methods.

2) While behavioral analysis can be made when these crayfish are on surface, nothing can be stated of their underground times. In this sense, it is an error to assign “inactivity” for underground times.

3) Discussion needs to be improved.

Comments:

- Study Site: More basic description is required of the study area around the filming spot, to better explore the results. It is said that most species (tertiary and secondary burrowing crayfish) inhabit lotic and lentic environments but it is not clear how far are the Primary burrowing species of this study from water bodies, for instance. It is said in the Introduction that “Lacunicambarus thomai is a burrowing crayfish species with a high propensity to inhabit burrows in marshes, roadside ditches, and flooded fields (38). Populations of L. thomai often live in localized colonies with conspecifics and inhabit burrows that are relatively simple but can nonetheless be up to 1-1.5 m deep (38,39)”. However, nothing, no information is provided of the specific area where the study took place: proximity from rivers, vegetation types and cover densities, average ranges of daily environmental factor variations, Latitudinal coordinates.

- Burrows were selected and filmed. How far is one burrow from the other? Inform the average land area inside which the 6 burrows were located. One individual is associated to each burrow – is that an assumption or are there evidences that they are solitary? Furthermore, it is said that burrows may have more than one entrance (line 67), how was this issue treated here?

- It is said in Statistical Analysis that none of the independent variables (humidity, temperature, and time) were strongly correlated (all r < 0.6). A representative graph depicting an average 24h variation of environmental factors in the studied season would be informative in Supplemental Material. How was time included in the models, as a categorical or continuous variable? A continuous variable with linear increase (such as a sequence from 0 to 23) would create artificial associations in the model. Please evaluate, based on the statistical parameters found in Table 3, if it is not enough to use a simple model without interaction between time, humidity and temperature to understand the influence of environmental factors on the surface activity. The reason for this question is that the complexity of the best-fit model seems to have inhibited any discussion about the analysis in the end.

- The Results section start with “Hourly Activity” (which should be “Hourly surface activity”) and Figure 4, but there is no explanation as to how this was calculated. It is explained in Statistical Analysis that for an individual to be considered active on surface in one specific hour and day, it needs to be seen in any time point within that hour in that day. Then, how was the group average/percentage calculated taking into account that each contributing individual was registered for a different number of days? Individuals that had more filmed days should not weight more. Finally, Legend Figure 4 needs to inform that averages were calculated taking into account all individuals and all days.

An explicit description of calculation should also be added to “percentage of time spent active throughout daytime” and “ proportion of time spent on surface”. In Table 5, how was the “mean duration” of each behavior calculated, taking into account individuals and number of days each individual was filmed?

In Figures 5, 6 and 7, it is interesting to show the “combined” proportions. However, it is biased by individual 2, which was filmed for more days. Could an unbiased calculation be made here?

In Figure 8 the number of observations is again biased to individual 2.

- Figure 5 and associated text: comparison should be between “surface” and “underground”, not between “active” and inactive” because nothing is known about what the crayfish are doing underground. The same for Figure 6: “nighttime on surface” and “daytime on surface”. In Line 281, replace “ active” by “on surface” in “The percentage of time that crayfish were active during the daytime”. In Line 283, “Regarding nighttime activity on surface”. Legend Figure 6: “percentage of time that each individual crayfish spent on surface during the day and the night”.

- Discussion: In contrast to Bearden et al. (2021), this study brings more information about the behavioral complexity of this particular crayfish species. However, the lengthy discussion is mainly descriptive of results. The authors should explore, for instance, what was found in statistical analysis and how could this be connected to the specificity of the studied environment, to take full advantage of the in situ study. Another suggestion is to take Bearden et al. (2021) as a reference, discuss how the results are constrained by the particular season and microhabitat that was covered in this study.

- Humidity was indicated as the most important factor modulating surface appearance in crayfish. This variable, as well as all others were collected from a meteorological station. Any thoughts about the validity of using only macro-environmental measurements in association with behaviors that are restricted to the spatial scale of the entrance of a burrow?

- Temperature was shown to be a strong predictor of surface activity. A strong suggestion for future studies is to also consider underground temperature in this analysis. For instance, it has been shown in endothermic subterranean rodents that a combination of external and underground temperatures predict better the episodes of surface emergences (Jannetti et al., Conservation Physiology 2019 v. 7, p. coz044). It is reasonable to assume that similar influence is potentially valid for these crayfish.

- This crayfish display clear postural signatures that enable behavioral identification through the relative position of body coordinates (Figure 3). This could potentially be used in future automated video analysis of activity patterns.

Minor Comments:

Fig.2C: view of the camera

Line 248: remove extra “were”

Line 319: “every single behavior was more likely to occur during day or night compared to another” An alternative could be “daily phase”

Table 4: what does this mean? “the activity of L. thomai was negatively related to the activity of crayfish.”

Reviewer #2: I understand that behaviour of strictly burrowing crayfish is difficult to study, so the information presented by the authors is surely interesting, and novel. However, the entire manuscript is based on only six individuals that were not even characterized. So, data are very preliminary and should be interpreted more cautiously. Behavioural categories should be also analysed together, considering that are dependent data. I am sorry not to be more positive, but a more specialised journal seems to be more appropriate for the manuscript.

Lines 50-52, 109: for the readers it would be interesting to specify that these species are North American

Line 67: maybe opening is more suitable than portal

Lined 128-130: so how many burrows were checked before selecting only six? How about the density of the burrows in the study area?

Line 132: crayfish could have been attracted out of the burrows with some baits after the footage to characterize them (for hunt behaviour it is reported they leave the burrow for example)

Line 182: were the behavioural data checked for normality?

Lines 218-222: those behaviours are dependent each other, so it is better to analyse them together because when crayfish are guarding, for example, they are not feeding or hunting

Line 235: please delete during the study

Line 270: please 5 not in italics

I suggest merging Fig 5 and 6 in one figure

Table 5: please report all the duration in seconds. Moreover, please change during with duration in the caption

Tables should be better drafted and presented

Line 343: please correct five

Line 345: please better rephrase this sentence

Line 356: Loughman et al. 2018 is not present in the bibliography; 46 is Loughman et al. 2015

Line 371, 448: please consider that only six individuals were observed, so I suggest being more cautious in this statement

Line 381: please do not use intricacies but richness or diversity

Line 415: please correct “is required”

Line 436: I think it is “use its claws to push the mud”

**********

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PLoS One. 2022 Oct 14;17(10):e0273540. doi: 10.1371/journal.pone.0273540.r002

Author response to Decision Letter 0

18 May 2022

Dear Dr. Junhu Dai,

Thank you for allowing us the opportunity to revise our manuscript. The comments from the two reviewers were extremely helpful and made the manuscript much stronger. Based on their suggestions, we incorporated most suggestions to our manuscript.

Below, you will find a letter detailing how we dealt with each comment of each reviewer. Where applicable, we state what we have changed and the location in which you will find our modifications in our updated manuscript.

We address each reviewer comment (C#) in individual answers (A#) to keep everything organized.

Reviewer #1:

C1: The authors made an interesting work, continuously filming the surface behavior of crayfish which were thought before to stay mostly or totally in underground burrows. They registered activity both during the day and night along with daily environmental factor variations and analyzed their behavior through the video recordings. While most behavioral studies in crayfish have been done in laboratory settings, this one contributes by rescuing ecological context and opening new perspectives of research. It is mostly well done but there are three general issues to be considered:

A1: Thank you for the generous comments. We appreciate your suggestions and we have attempted to address all your comments below.

______________________________________________________________________________

C2: One of the big contributions of this work is to enable discrimination of individual behavior, which is not a simple task in natural observations. Individual data presentations are fine but I suspect that when average analysis are presented, some or most of them are not considering that one individual (#2) with more than double the number of data collection is biasing the results. This can be deduced from some graphs while others do not let us know because description of average calculations are missing in Materials and Methods.

A2: We agree that analyzing behavioral data presents several issues and that this issue can be exacerbated by having an unequal sample size (263 hrs of footage for crayfish 2 vs 40 hours for crayfish 1). This is why we have chosen to present the majority of our results in terms of the percentages and not raw values. Reporting this information in terms of the raw values would certainly bias our results, as you suggest. However, by reporting this information (Figures 5, 6, and 7) in percentages, we believe that this is the most appropriate way to report our results and adjust for the bias. Furthermore, our manuscript reports relatively few true statistical analyses (and instead opts for more descriptive statistics of what we observed). This aligns with the primary goals of our manuscript—to describe the behavioral diversity of this elusive crayfishes and demonstrate how this new methodology can be lucrative in the field of crustacean behavioral ecology.

______________________________________________________________________________

C3: While behavioral analysis can be made when these crayfish are on surface, nothing can be stated of their underground times. In this sense, it is an error to assign “inactivity” for underground times.

A3: We agree. Our manuscript can only report of the surface activity, and not any potential underground behaviors. We have altered our language throughout the entire manuscript to account for this comment. We have changed any discussion of “inactivity” to “inactivity at the surface”.

____________________________________________________________________________

C4: Discussion needs to be improved.

A4: We have taken several of your comments regarding topics that require additional discussion and expanded on them.

______________________________________________________________________________

C5: Study Site: More basic description is required of the study area around the filming spot, to better explore the results. It is said that most species (tertiary and secondary burrowing crayfish) inhabit lotic and lentic environments but it is not clear how far are the Primary burrowing species of this study from water bodies, for instance. It is said in the Introduction that “Lacunicambarus thomai is a burrowing crayfish species with a high propensity to inhabit burrows in marshes, roadside ditches, and flooded fields (38). Populations of L. thomai often live in localized colonies with conspecifics and inhabit burrows that are relatively simple but can nonetheless be up to 1-1.5 m deep (38,39)”. However, nothing, no information is provided of the specific area where the study took place: proximity from rivers, vegetation types and cover densities, average ranges of daily environmental factor variations, Latitudinal coordinates.

A5: We agree that information on our study site was lacking in the original version of the manuscript. We have now included a detailed description of the location in which we conducted this study (see Lines 127-134). Because our sampling location is on a residential property, we choose to not report exact coordinates.

______________________________________________________________________________

C6: Burrows were selected and filmed. How far is one burrow from the other? Inform the average land area inside which the 6 burrows were located. One individual is associated to each burrow – is that an assumption or are there evidences that they are solitary? Furthermore, it is said that burrows may have more than one entrance (Line 67), how was this issue treated here?

A6: We have updated this information within our manuscript. We now provide information regarding how far away one burrow was from one another (Lines 131-132), as well as our assumption that each burrow was occupied by a single crayfish (Lines 141-144). We also clarify how we dealt with the issue with burrows with more than a single entrance (Lines 151-153).

______________________________________________________________________________

C7: It is said in Statistical Analysis that none of the independent variables (humidity, temperature, and time) were strongly correlated (all r < 0.6). A representative graph depicting an average 24h variation of environmental factors in the studied season would be informative in Supplemental Material. How was time included in the models, as a categorical or continuous variable? A continuous variable with linear increase (such as a sequence from 0 to 23) would create artificial associations in the model. Please evaluate, based on the statistical parameters found in Table 3, if it is not enough to use a simple model without interaction between time, humidity and temperature to understand the influence of environmental factors on the surface activity. The reason for this question is that the complexity of the best-fit model seems to have inhibited any discussion about the analysis in the end.

A7: We have now included 4 new figures in the supplemental materials (Figures S1, S2, S3, and S4) which display the relevant environmental data during our study period.

In the original manuscript, we reported that there were no strong statistical correlations between humidity, temperature, and time (as you mentioned). In our environmental analysis, time was coded as a continuous variable. We tested for the correlations beforehand to potentially deal with any artificial associations that you hint at. However, because of the low correlation values, we decided to proceed with using time as a continuous variable. Furthermore, the fact that time is not a strong predictor of activity (Table 4) suggests that there is no underlying association between these variables.

Our best fit model was quite complex, as you note. It was a model with 3 single parameters, and two interaction parameters. This why is we not only conducted a model-selection procedure (results in Table 3), but also the multi-model average procedure (results in Table 4). The results from our full model averaging technique allow us to evaluate all of the models together and construct effect sizes and weights for each of the terms in our model. This is why we come to the conclusion that humidity had the strongest overall effect (Table 4) compared to the other models. We have now included a better discussion of this result in our discussion section (see A14).

______________________________________________________________________________

C8: The Results section start with “Hourly Activity” (which should be “Hourly surface activity”) and Figure 4, but there is no explanation as to how this was calculated. It is explained in Statistical Analysis that for an individual to be considered active on surface in one specific hour and day, it needs to be seen in any time point within that hour in that day. Then, how was the group average/percentage calculated taking into account that each contributing individual was registered for a different number of days? Individuals that had more filmed days should not weight more. Finally, Legend Figure 4 needs to inform that averages were calculated taking into account all individuals and all days.

A8: We have changed this section to “Hourly Surface Activity” as you suggest.

We have clarified how this was calculated in the methods sections (Lines 201-209). Again, we report these values in terms of percentages, and not the raw number of hours each crayfish was active to avoid any potential sampling biases as you suggest.

We have also updated the legend for Figure 4 to include those averages were calculated by taking into account for all individuals and all days throughout our study.

______________________________________________________________________________

C9: An explicit description of calculation should also be added to “percentage of time spent active throughout daytime” and “ proportion of time spent on surface”. In Table 5, how was the “mean duration” of each behavior calculated, taking into account individuals and number of days each individual was filmed?

A9: We have updated our methods to also include information as to how we calculated these percentages. By reporting these values in percentages, we avoid any issues based on the number of hours each crayfish was sampled and filmed.

We have updated the legend in figure 5 to describe that our mean durations were calculated based on all crayfish. This data is reported to be entirely descriptive in nature, and therefore we believe that reporting such means are the most informative way to describe these different behaviors.

______________________________________________________________________________

C10: In Figures 5, 6 and 7, it is interesting to show the “combined” proportions. However, it is biased by individual 2, which was filmed for more days. Could an unbiased calculation be made here?

A10: Reporting our results in terms of percentages is the best way to provide an unbiased calculation for these figures, which are primarily meant to be descriptive and exploratory in nature. If we would have reported the data from the raw amount of time that each behavior/activity was performed, then this would certainly be biased. This is why we chose to reported the data in terms of percentages throughout the manuscript.

______________________________________________________________________________

C11: In Figure 8 the number of observations is again biased to individual 2.

A11: Yes, Figure 8 (but not 5, 6, and 7) is biased based on the increased number of observations from individual 2. This figure (and the analysis in general) are describing trends in whether or not a specific behavior was more or less likely to occur at day versus night. We have included information regarding this issue in our manuscript (Lines 201-203). Anecdotally, if you look at the percentages of all behaviors reported in Fig 5, 6, and 7, there is a similar degree of surface activity/behaviors being exhibited by each crayfish, which implies that this bias may be minimally impacting our results.

______________________________________________________________________________

C12: Figure 5 and associated text: comparison should be between “surface” and “underground”, not between “active” and inactive” because nothing is known about what the crayfish are doing underground. The same for Figure 6: “nighttime on surface” and “daytime on surface”. In Line 281, replace “ active” by “on surface” in “The percentage of time that crayfish were active during the daytime”. In Line 283, “Regarding nighttime activity on surface”. Legend Figure 6: “percentage of time that each individual crayfish spent on surface during the day and the night”.

A12: You are correct; we do not want to mislead the readers. Now, all of our figures have been accounted for the fact that we are only reporting surface activity and that we cannot comment on underground. Activity. Figures 5 and 6 are now updated accordingly. We have also changed the legend for Figure 6

______________________________________________________________________________

C13: Discussion: In contrast to Bearden et al. (2021), this study brings more information about the behavioral complexity of this particular crayfish species. However, the lengthy discussion is mainly descriptive of results. The authors should explore, for instance, what was found in statistical analysis and how could this be connected to the specificity of the studied environment, to take full advantage of the in situ study. Another suggestion is to take Bearden et al. (2021) as a reference, discuss how the results are constrained by the particular season and microhabitat that was covered in this study.

A13: We agree that we could have expanded on our discussion section. Based on many of your suggestions below we believe that the discussion has significantly improved.

______________________________________________________________________________

C14: Humidity was indicated as the most important factor modulating surface appearance in crayfish. This variable, as well as all others were collected from a meteorological station. Any thoughts about the validity of using only macro-environmental measurements in association with behaviors that are restricted to the spatial scale of the entrance of a burrow?

A14: We agree that the macro environmental variables that we relate to surface activity are far from ideal for such studies. We have updated our discussion to point this out and suggest that future studies take into account the variables potential micro-environmental influences within and around the burrow entrances (Lines 406-422).

______________________________________________________________________________

C15: Temperature was shown to be a strong predictor of surface activity. A strong suggestion for future studies is to also consider underground temperature in this analysis. For instance, it has been shown in endothermic subterranean rodents that a combination of external and underground temperatures predict better the episodes of surface emergences (Jannetti et al., Conservation Physiology 2019 v. 7, p. coz044). It is reasonable to assume that similar influence is potentially valid for these crayfish.

A15: This is a great suggestion to add to our discussion section. We have now included that looking at the underground temperature variation is a fruitful area for future directions (Lines 416-422).

______________________________________________________________________________

C16: This crayfish display clear postural signatures that enable behavioral identification through the relative position of body coordinates (Figure 3). This could potentially be used in future automated video analysis of activity patterns.

A16: That is a great suggestion to add the discussion. We have included these postural changes to be used in the identification of automated video analysis in the future (Lines 511-513).

______________________________________________________________________________

C17: Fig.2C: view of the camera

A17: We have edited this sentence accordingly.

______________________________________________________________________________

C18: Line 248: remove extra “were”

A18: We have removed the extra “were”

______________________________________________________________________________

C19: Line 319: “every single behavior was more likely to occur during day or night compared to another” An alternative could be “daily phase”

A19: We believe that the current wording provides for clarity because “daily phase” may be confused with “day”.

______________________________________________________________________________

C20: Table 4: what does this mean? “the activity of L. thomai was negatively related to the activity of

crayfish.”

A20: This was a typo and should have read “Thus, the activity of L. thomai was negatively related to the degree of environmental humidity.” We have made this change to the Table 4 text. Thank you.

______________________________________________________________________________

Reviewer #2:

C21: I understand that behaviour of strictly burrowing crayfish is difficult to study, so the information presented by the authors is surely interesting, and novel. However, the entire manuscript is based on only six individuals that were not even characterized. So, data are very preliminary and should be interpreted more cautiously. Behavioural categories should be also analysed together, considering that are dependent data. I am sorry not to be more positive, but a more specialised journal seems to be more appropriate for the manuscript.

A21: We agree that the fact that our study being only conducted on six individuals is a limitation to our study. Low sample sizes are typical for this type of work, because of the time intensive nature and complexities of naturalistic studies (see 6 individuals reported in Janettii et al. 2019) Although this work was only conducted on six individuals (that have unknown demographic information), we believe that the amount and nature of our data is worthy of publication.

In the previous and updated versions of our manuscript, we attempt not to overstate our results as they are preliminary and exploratory in nature. Furthermore, although we do not report the demographic data from these crayfishes (sex, etc), there is no data that has alluded to sex differences in burrowing crayfish behavior. We do agree that this is an interesting and important angle for future studies though, so we have included that this information should be investigated in the future (Lines 507-508).

Despite these limitations, we still believe that we provide a novel methodology and interesting results (which reviewer 1 highlights) that will be of interest to a wide audience. Because the scope of PLOS ONE is to publish papers based on their scientific validity and methodology, we believe that our study is of interest to an audience outside of smaller, taxonomic focused journals.

We respond to the comment on behavioral categories being analyzed together below (see A27).

______________________________________________________________________________

C22: Lines 50-52, 109: for the readers it would be interesting to specify that these species are North American

A22: We have edited Lines 50-52 to clarify that these are North American species (Line 50-51)

______________________________________________________________________________

C23: Line 67: maybe opening is more suitable than portal

A23: Portal is a term burrowed from the literature on mammalian burrowing behavior and is widely used to refer to crayfish burrow openings. Therefore, we prefer to keep this language consistent with prior published papers. We have included a few examples below.

Glon, M. G., Adams, S. B., Loughman, Z. J., Myers, G. A., Taylor, C. A., & Schuster, G. A. (2020). Two new species of burrowing crayfish in the genus Lacunicambarus (Decapoda: Cambaridae) from Alabama and Mississippi. Zootaxa, 4802(3), 401-439.

Loughman, Z. J. (2010). Ecology of Cambarus dubius (upland burrowing crayfish) in north-central West Virginia. Southeastern Naturalist, 9(sp3), 217-230.

______________________________________________________________________________

C24: Lined 128-130: so how many burrows were checked before selecting only six? How about the density of the burrows in the study area?

A24: We have now included much more information regarding our study location. Based on the relatively small number of burrows at the study location, we choose to only focus on a small sample size but to collect as much data as possible on each of these adult individuals.

______________________________________________________________________________

C25: Line 132: crayfish could have been attracted out of the burrows with some baits after the footage to characterize them (for hunt behaviour it is reported they leave the burrow for example)

A25: Yes, that is true. Unfortunately, we did not capture these individuals. We have included information how future studies need to explore how different demographics may exhibit such behaviors differently (lines 50-51).

______________________________________________________________________________

C26: Line 182: were the behavioural data checked for normality?

A26: Yes, all data and model fits were checked for normality. We have now included this information in the manuscript (Lines 197-199).

______________________________________________________________________________

C27: Lines 218-222: those behaviours are dependent each other, so it is better to analyse them together because when crayfish are guarding, for example, they are not feeding or hunting

A27: This comment highlights the complexities of working with behavioral data, because an organism cannot perform more than a single behavior at once. Therefore, this is why we chose to primarily focus on broad, descriptive statistics throughout our study. Because of the novelty of these findings and the potential impact on our understanding of crustacean behavioral ecology, we believe that this is the proper way to analyze report our data at this stage.

______________________________________________________________________________

C28: Line 235: please delete during the study

A28: We have deleted this phrase.

______________________________________________________________________________

C29: Line 270: please 5 not in italics

A29: We have unitalicized this number.

______________________________________________________________________________

C30: I suggest merging Fig 5 and 6 in one figure

A30: We prefer to keep these figures separate as to avoid confusion between the messages of figure 5 (observed at surface vs. underground) and figure 6 (observed at surface during the day versus observed at surface during the night).

______________________________________________________________________________

C31: Table 5: please report all the duration in seconds. Moreover, please change during with duration in the caption

A31: We have changed the table to report each behavior in. We have changed during to duration as you suggested.

______________________________________________________________________________

C32: Tables should be better drafted and presented

A32: Without any comments on the issues of the tables, we are unsure what to change base.

______________________________________________________________________________

C33: Line 343: please correct five

A33: We change made this change.

______________________________________________________________________________

C34: Line 345: please better rephrase this sentence

A34: We have rephrased this sentence accordingly (Lines 366-368).

______________________________________________________________________________

C35: Line 356: Loughman et al. 2018 is not present in the bibliography; 46 is Loughman et al. 2015

A35: Thank you for noticing this issue. We meant Loughman et al. 2015 and we have made this change.

______________________________________________________________________________

C36: Line 371, 448: please consider that only six individuals were observed, so I suggest being more cautious in this statement

A36: We agree that we need to be more cautious with these statements. We have added additional information to these sections based on your comments (see Lines 394-395 and Lines 494-495).

______________________________________________________________________________

C37: Line 381: please do not use intricacies but richness or diversity

A37: We have changed the work intricacies to richness as you have suggested.

______________________________________________________________________________

C38: Line 415: please correct “is required”

A38: We have changed “are required” to “is required”

______________________________________________________________________________

C39: Line 436: I think it is “use its claws to push the mud”

A39: Thank you for catching this error. We have fixed the typo.

Attachment

Submitted filename: response_to_reviewers_1.docx

Click here for additional data file.^{(33.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0273540.r003

Decision Letter 1

Louis-Felix Bersier

19 Jul 2022

PONE-D-21-39709R1On the surface or down below: Field observations reveal a high degree of surface activity in a burrowing crayfish, the Little Brown Mudbug (Lacunicambarus thomai)PLOS ONE

Dear Dr. Diehl,

Since I had difficulties finding a second Reviewer, I decided to review your revised manuscript myself. As you will see, the negative Reviewer of your first submission appreciated your corrections and judged that you contribution is now ready for publication. However, I found some problems that need to be settled before your paper is accepted. The main point concerns difficulties to link the results given in the text with those in the figures. I think that there may be some mistakes. For this reason, please carefully check your Result section. Also, I made several editorial recommendations. You will find all this information in the attached file "D-21-39709_R1_LFB.pdf". I appreciated your work and think that it will be a useful contribution to crayfish biology.

Please submit your revised manuscript by Sep 02 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Louis-Felix Bersier, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

See attached file "D-21-39709_R1_LFB.pdf"

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: I appreciate the responses and corrections provided by the authors, I do not have further comments to be addressed

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

**********

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PLoS One. 2022 Oct 14;17(10):e0273540. doi: 10.1371/journal.pone.0273540.r004

Author response to Decision Letter 1

6 Aug 2022

Dear Dr. Louis-Felix Bersier,

Thank you for allowing us the opportunity to revise our manuscript. Your comments were extremely helpful and made the manuscript much stronger. Based on their suggestions, we incorporated most suggestions to our manuscript. Below, you will find a letter detailing how we dealt with each comment you provided.

We address each comment (C#) in individual answers (A#) to keep everything organized.

Editor Comments:

C1: This level of precision is not necessary. I recommend rounding at unity here (21% to 69%)

A1: We have rounded these percentages.

______________________________________________________________________________

C2: Could be abridged as "tertiary-, secondary-, and primary burrowing species"

A2: This is a good suggestion, we have changed the text accordingly.

______________________________________________________________________________

C3: delete

A3: We have deleted the words “the setup of” here.

______________________________________________________________________________

C4: I guess "... unable to report ..."

A4: Yes, you are correct. We have edited the text here.

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C5: Meaning not clear to me. If it indicates that this behavior occurs mostly during the night, it should not be part of the description in the table, but should be stated in the results.

A5: This statement (more exposed during the night) has been deleted because we agree that it is unclear. We meant to mean that when the crayfish exhibit the guard behavior during the night, they are typically more exposed out of their burrow portal. But we did not quantify this, so we chose to delete it from the text.

______________________________________________________________________________

C6: The images are very useful and raise a question since these two behaviors are very similar. As a non-specialist, I would like some words (and possibly a reference) that explain and justify this distinction.

A6: After some discussion, we agree with all of your comments regarding our denotation of these behaviors of “guard” and “rest” behaviors. We now refer to both of these behaviors as “rest” and separate them based on “rest-claws open/rest - open” and “rest-claws joined/rest – joined”. Although rest – closed may represent a guarding behavior, we believe that because this work is preliminary, we are keeping the language simple. The manuscript and figures have been changed accordingly. We believe that this language will properly describes the behaviors of the crayfishes would making any presumptions on the function of the posture.

______________________________________________________________________________

C1: There may be an unimodal relationships with these variable, reflecting optima. Did you check for this possibility ?

A1: We did check for this, but because there was not as much variation in the environment variables, we did not find any optimum relationship between activity and such environmental variables.

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C1: Personally, I would place the Tables 3 and 4 in the Supporting Information (only a suggestion).

A1: We have moved Table 3 and Table 4 to the SI as you have suggested.

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C1: This should be part of the Method section, e.g., on line 216, in the parenthesis "(

A1: We have moved this section the methods section as you suggested.

______________________________________________________________________________

C1: Same remark as for the Abstract.

A1: All of our percentage values have been adjusted to be reported in the way that you suggest.

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C1: I do not see the correspondance between the percentages in the text and in Fig. 5. Please check if it is correct or provide better explanations (are the colors reversed in the figure ?)

A1: Thank for noticing this mistake. Yes, the colors on the figure legend should be reversed. in Figure 5. We have fixed this accordingly. Now the numbers and percentages add up correctly.

______________________________________________________________________________

C1: Again, I have problem with the values in the text and in Table 5. Crayfish 4 is the only one that foraged during the day. From Table 5, this happened only once, with a total of 360s, which is clearly not equal to 0.34 hr. Please explain.

A1: After checking back at our data, you are correct, we mistakenly calculated a larger percentage for the daytime activity of Crayfish 4. This change is now reflected in the manuscript and in the Figure 7, and Table 5. We re-checked the other crayfish as well and the results are correct for them.

______________________________________________________________________________

C1: Same remark as for Tables 3 and 4. The results are different but redundant with Fig. 7.

A1: We have moved this Table (Table 5) to the SI as you have suggested.

______________________________________________________________________________

C1: But Fig. 7 shows the opposite pattern! Table 5 support this.

A1: Thank you for catching this error. This was a typo and now correctly states that “The majority of their time was spent in the relax position during the night, and relaxing was more likely to occur during the night compared to during the day.”

______________________________________________________________________________

C1: I think that "proportion" is more adequate here.

A1: We have reworded this to proportion based on your suggestion.

______________________________________________________________________________

C1: Again, I would place this Table in the SI, as the main information can be visualized in Fig. 8.

A1: We have moved this table to the SI based on your suggestions.

______________________________________________________________________________

C1: This relates to my question concerning Fig. 3 (line 183). Does reference 51 separates these two behaviors based on claws' position?

A1: See A6.

______________________________________________________________________________

C1: At this point, I am uncomfortable with your choice of words (relax and guard) for the two behaviors based on position of claws. Perhaps "guard - claws open" and "guard - claws joined" may be more appropriate?

A1: See A6.

______________________________________________________________________________

Attachment

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PLoS One. doi: 10.1371/journal.pone.0273540.r005

Decision Letter 2

Louis-Felix Bersier

11 Aug 2022

On the surface or down below: Field observations reveal a high degree of surface activity in a burrowing crayfish, the Little Brown Mudbug (Lacunicambarus thomai)

PONE-D-21-39709R2

Dear Dr. Diehl,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Thank you for your thorough consideration of my comments. I went through your corrected version and found that it is ready for publication. I just noted a probable mistake on lines 243-244: "(all r > 0.6)" rather than "(all r < 0.6)". Please check this before submitting your final document.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0273540.r006

Acceptance letter

Louis-Felix Bersier

2 Sep 2022

PONE-D-21-39709R2

On the surface or down below: Field observations reveal a high degree of surface activity in a burrowing crayfish, the Little Brown Mudbug (Lacunicambarus thomai)

Dear Dr. Diehl:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Recording of an excavation observation during the night.

(MP4)

Click here for additional data file.^{(15.8MB, mp4)}

S2 File. Recording of a foraging observation during the night.

(MP4)

Click here for additional data file.^{(87.5MB, mp4)}

S3 File. Recording of a hunt observation during the night.

(MP4)

Click here for additional data file.^{(45.4MB, mp4)}

(DOCX)

Click here for additional data file.^{(14.2KB, docx)}

S2 Table. Coefficients and standard errors (SE) for the model predicting the activity of L. thomai based on full model averaging.

(DOCX)

Click here for additional data file.^{(13.4KB, docx)}

S3 Table. The number of observations and the mean duration of each behavior observed during our study.

(DOCX)

Click here for additional data file.^{(13.7KB, docx)}

S4 Table. Results from Chi-squared test for the number of behaviors that occur during the day and versus the night.

(DOCX)

Click here for additional data file.^{(13.4KB, docx)}

S1 Fig. Average, minimum, and maximum air temperature at our study location in Hickory, PA throughout our study period (June 2020 –July 2020).

(PNG)

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S2 Fig. Average, minimum, and maximum humidity at our study location in Hickory, PA throughout our study period (June 2020 –July 2020).

(PNG)

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S3 Fig. Average, minimum, and maximum air pressure at our study location in Hickory, PA throughout our study period (June 2020 –July 2020).

(PNG)

Click here for additional data file.^{(577.4KB, png)}

S4 Fig. Precipitation at our study location in Hickory, PA throughout our study period (June 2020 –July 2020).

(PNG)

Click here for additional data file.^{(283.9KB, png)}

Attachment

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Data Availability Statement

The data are available at Dryad at the following DOI: https://doi.org/10.5061/dryad.kh189328r.

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PERMALINK

On the surface or down below: Field observations reveal a high degree of surface activity in a burrowing crayfish, the Little Brown Mudbug (Lacunicambarus thomai)

Kaine M Diehl

Nicoleena M Storer

Hogan D Wells

Destinee A Davis

Zachary J Loughman

Zackary A Graham

Roles

Abstract

Introduction

Fig 1. Photographs of adult burrowing crayfish, the Little Brown Mudbug, Lacunicambarus thomai, exhibiting daylight activity outside of its burrow.

Methods

Study site

Videography

Fig 2. Images representing our custom video surveillance mount.

Table 1. Dates and total time that each crayfish was filmed throughout the duration of our study.

Ethogram

Table 2. Ethogram used to quantify the behaviors from recordings of Lacunicambarus thomai.

Fig 3. Example of rest–claws openand rest–claws joined behaviors from video surveys of Lacunicambarus thomai.

Environmental data

Statistical analysis

Results

Hourly surface activity

Fig 4. The percent of Lacunicambarus thomai (n = 6) that were active for each hour during our study.

Environmental predictors of surface activity

Surface activity

Fig 5. The percentage of time that Lacunicambarus thomai spent visible at the surface of their burrow (active at surface) or not visible underground in their burrow (underground).

Day vs. night surface activity

Fig 6. The percentage of time that Lacunicambarus thomai was observed exhibiting surface activity during the day and during the night.

Fig 7.

Fig 8. Mosaic plot of the behavior exhibited by each crayfish (excavate, forage, guard, hunt, relax, under) based on the time of day it was recorded (night, day).

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Junhu Dai

Roles

Author response to Decision Letter 0

Decision Letter 1

Louis-Felix Bersier

Roles

Author response to Decision Letter 1

Decision Letter 2

Louis-Felix Bersier

Roles

Acceptance letter

Louis-Felix Bersier

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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