DOI: 10.5061/dryad.34tmpg4qr
title: Rising surface temperatures lead to more frequent and longer burrow retreats in males of the fiddler crab, Minuca pugnax

description: The fiddler crab Minuca pugnax occupies thermally unstable mudflat
 habitats along the eastern United States coastline, where it uses
 behavioral thermoregulation, including burrow retreats, to manage body
 temperature (Tb). We explored the relationship between frequency of burrow
 use and environmental conditions, including burrow and surface
 temperatures, relative tidal height, and time of day, by twenty male M.
 pugnax in breeding areas around Flax Pond, New York, USA. We found a
 highly significant positive correlation between burrow use and surface
 temperature, with a clear shift to longer times underground above 32°C
 degrees. We also experimentally heated live crabs in the laboratory and
 allowed them to retreat into cooled artificial burrows while continuously
 measuring body temperatures (Tb). Laboratory data on cooling times were
 compared to field observations of burrow retreat durations. The median
 burrow stay in the field of 2.74 min was enough time for our laboratory
 crabs to capture over 70% of the cooling potential of artificial burrows
 10 or 15 degrees below Tb. Because crab bodies in burrows experience
 exponential declines in Tb due to Newton’s law of cooling, there are
 diminishing returns to remaining in a burrow, and many crabs probably
 leave before coming to equilibrium. For M. pugnax, burrow retreats reduce
 time spent feeding and courting, activities that only occur on the
 surface. Current concerns about the impacts of climate change on animals
 include whether compensatory mechanisms, like more frequent and longer
 burrow retreats, will come at the cost of other behaviors necessary for
 survival and reproduction.
description: Field-based observations of burrow
 retreats and environmental temperatures The<em>
 </em>burrow retreats of displaying <em>Minuca
 pugnax</em> males were observed in the marsh by the Flax Pond Marine
 Laboratory in Old Field, New York, USA (40.9633274,-73.1439265) on an open
 area of exposed sediment in a location otherwise dominated by
 <em>Spartina alterniflora</em> and <em>Iva
 frutescens</em>. During the study period, which began
 on a neap tide in June 2022, the area was not inundated during the daily
 tidal cycle. Video data were collected with a GoPro Hero5 camera mounted
 on a tripod with a boom arm that extended horizontally over the study
 area. Eleven recordings (1 to 3 per day) lasting between 54 and 130
 minutes were made between June 7–12, 2022. Crabs were recorded throughout
 the tidal cycle during daylight hours between 08:45 and 15:40, although
 the durations varied between sampling days. From these recordings, 23
 focal crabs (4–6 per day) were selected and scored for burrow retreat
 behaviors. Focal crabs, which were chosen based on a quick scan of the
 videos before any scoring took place, were individual burrow owners deemed
 likely to stay in view (e.g., not obscured by vegetation or near the edge
 of the field of view). We assumed that burrow owners seen in different
 video segments for the same day were the same crab and that different
 crabs were filmed each day as the camera was moved to different locations
 in the same general area. The assumption of consistent burrow occupancy
 was supported by the fact that we never saw a focal crab give up a burrow
 and leave the area during 870 total minutes of video segments. Two Tidbit
 temperature loggers (Onset model UTBI-001) were placed within 0.5 m of the
 area where crabs were video recorded each day. One was at the bottom of an
 artificial burrow made of an open-ended PVC tube with a diameter of 5 cm,
 hammered to a depth of 25 cm, and the other was on the surface of the
 substratum adjacent to the artificial burrow. Artificial burrows were used
 to obtain temperatures at a consistent depth, which would have been too
 difficult with crab-constructed burrows, which vary in pitch and
 morphology. Temperature measurements were collected at one-minute
 intervals continuously throughout the
 day.<br> For each video segment, focal crab
 scoring began 10 minutes into the recording to allow for a return to
 normal behavior following disturbances caused by a camera setup or a
 battery change. Using an instantaneous sampling approach, we scored
 whether each focal crab was in or out of its burrow at 1-min intervals
 following the acclimation period. Additionally, we recorded the durations
 of burrow retreats for each focal animal. A burrow retreat was scored if
 the entire crab disappeared from view, with the time of the retreat
 beginning when half of the crab’s body was inside the burrow and ending
 when half of its body had emerged. This approach was used because crabs
 exiting their burrows sometimes paused at the entrance, with the distal
 parts of some walking legs still inside. Waiting for the entire body to
 emerge would have artificially increased our estimate of the burrow
 duration. Three types of burrow retreats were
 observed during this study: retreats following a chase by a con- or
 heterospecific male (another fiddler species,<em> Leptuca
 pugilator</em>), retreats related to courtship where the focal crab
 entered its burrow and a female followed, and retreats that were not
 initiated by chases or potential mates. We only included the latter type
 in the analysis (n = 229) because they were more likely related to
 thermoregulation. Although courtship and aggression-related retreats
 (n=13) might have turned into thermoregulation-related retreats, they were
 usually short, often less than 30 s, and appeared to be relatively shallow
 (none of the females we observed entering a focal male’s burrow remained
 there long enough for mating to have occurred). We did not observe any
 retreats associated with burrow construction or potential predators.  
 Statistical analysis for
 field-based observations To explore how
 environmental conditions and time of day impacted the frequency of burrow
 retreats, we fit our data to a binomial logistic linear mixed model using
 the glmmTMB package in R, where burrow occupancy (in versus out) was the
 binary response variable. Sampling day and individual crab were coded as
 random effects, while surface temperature, burrow temperature, time of
 day, and relative tidal height (scaled between 0 and 10) were fixed
 effects. Tidal height was estimated using the NOAA tide station water
 level data from Port Jefferson (station 8514560), with a lag time of 1.5
 hours added (Flax Pond Marine Laboratory curator Stephen Abrams, pers.
 comm.). Relative tidal height was included as a variable even though the
 site remained exposed throughout the study because fiddler crabs show
 lunar periodicity for reproductive behaviors (Christy 1982, Morgan and
 Christy 1995). Hence, the timing of high and low tides might affect
 burrow-related behaviors.  In the original data set, each crab was
 recorded as either in or out of its burrow for each minute of sampling,
 resulting in 3727 total observations. However, including all data points
 led to a highly unstable model with an autocorrelation coefficient of 1.0,
 probably because crabs in burrows at one observation point were likely to
 still be there at the next one, as were crabs on the surface. To address
 the high serial correlation, binary data were combined into 20-min
 increments for each crab's time series, where the response variable
 became the number of times the crab was in the burrow for each increment.
 Time of day, tidal height, surface temperature, and burrow temperature
 were then averaged for each of these increments. This change resulted in a
 stable model with 196 observations and an autocorrelation of 0.75. The
 autoregression process (AR1) was applied to observations within crabs.
 Random intercepts were used to model the correlation among observations
 incurred by the hierarchical nature of the data, i.e., crabs were sampled
 within sampling day, and observations were clustered within crabs.
 Finally, we normalized all variables with the scale() function in R to
 avoid numerical instability when looking for interaction effects.
 To determine if duration of burrow
 retreats increased with increasing surface temperatures, we calculated
 median time underground and median surface temperature experienced for
 each focal crab and performed a Spearman correlation analysis. Also, upon
 discovering a behavioral shift around 30–32 °C, above which focal crabs
 were more likely to be underground, we performed a post hoc analysis,
 comparing the median burrow retreat durations above and below this
 temperature with a Wilcoxon Rank Sum Test. These analyses were performed
 in R with the ggpubr package. Finally, crab body sizes were measured from
 video images with ImageJ by comparing crab carapace width to a 2 cm ruler
 that was placed on the substratum within the crab
 habitat.  Laboratory-based cooling
 experiments Around 20 <em>Minuca pugnax
 </em>males were collected from Wareham, Massachusetts, USA
 (41.7587424, -70.7148547) on five different days between June 3 and July
 5, 2021, for the burrow cooling experiment and two additional
 investigations not described here. Crabs were brought to a laboratory at
 Mount Holyoke College (South Hadley, Massachusetts), where approximately
 ten individuals each were kept in covered plastic containers (27 x 20 x 13
 cm) partly filled with mud and water from the collection site, in addition
 to other natural items like rocks, shells, and seagrass. Because crabs
 were observed to deposit feed on the exposed mud inside their containers,
 we did not supplement their food. Crabs were selected at random for each
 treatment of the burrow cooling experiment and used within a week of
 collection.  For all treatments in the burrow
 cooling experiment, we heated individual crabs to a body temperature
 (T<sub>b</sub>) of around 35 °C by placing them on damp fine
 white marine sand (Clifford W. Estes Company, NJ) in a glass container
 warmed by a hot plate, while simultaneously heating them from above with a
 250-watt heat lamp bulb. While 35 °C is warmer than they prefer (Hews et
 al. 2021), we commonly find crabs in the field with this
 T<sub>b</sub>, and crabs warmed to this temperature in the
 laboratory did not appear to be impaired. Once a T<sub>b</sub>
 of around 35 °C was recorded (see below), crabs were allowed to enter an
 artificial burrow cooled to 30, 25, or 20 °C (n=10 for each temperature).
 Burrow temperatures of around 20-25 °C are within the range that we have
 seen for deeper burrows (25-30 cm) in the field, and
 30°C could be
 experienced near the top of a burrow, closer to the surface.
 The burrow was constructed using a 15 mL plastic centrifuge
 tube with a 1.5 cm diameter (Falcon) to create a hole in damp fine marine
 sand that filled a beverage cooler (YJ Home). T<sub>b</sub>
 was measured continuously (two measurements per 0.01s) using a flexible
 Type T microprobe connected to a Thermes USB Temperature Acquisition
 system (Physitemp Instruments, NJ).  The microprobe was inserted through a
 hole in the crab’s carapace into the branchial chamber and reversibly
 affixed to the carapace with a 50:50 pine rosin (Velesco) and beeswax
 (Stakich Inc., MI) mixture melted by a dental wax carving tool (SJK Lab,
 model J0801). Crabs were heated to the target temperature immediately
 following the probe insertion and then allowed to enter the burrow. Once
 crabs cooled to the target temperature, they were retrieved from the
 burrow, and the microprobe was removed. Crabs survived this procedure, and
 most were released at the collection site. Cooling curves
 for each temperature showing average T<sub>b</sub>s and 95%
 confidence intervals were constructed with the seaborn package version
 0.12.1 in Python 3. Crab body sizes were determined by measuring carapace
 width with a digital caliper.
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