Living on the Edge and Beyond of Anoxia: Evolutionary Ecological Insights From Inside Craysh Burrows

Burrowing is a common trait among crayfish thought to help species deal with adverse 40 environmental challenges. Here we used in-vivo experimental data and in-silico modelling of 41 oxygen saturation in a virtual burrow inhabited by crayfish. Except for the entrance 200 mm 42 region, the burrow microenvironment becomes anoxic, on average, within 8 hours, and 12-hour 43 day-night multiple cycles were not sufficient for refreshing the burrow microenvironment even 44 with temporary lack of crayfish. We asked whether the ecological category of crayfish 45 burrowing activity is reflected in the physiological ability to cope with hypoxia and anoxia. As 46 dissolved oxygen declined, respiration patterns of primary burrowers differed from those of 47 secondary and tertiary burrowers, showing also the highest variability in anoxia tolerance. 48 Secondary burrowers showed consistent tolerance with all species exhibiting a mean survival 49 of > 3h anoxic conditions. Tertiary burrowers were variable, exhibiting moderate to zero 50 tolerance of anoxia. The adaptive mechanisms to cope with hypoxia might be a basal legacy 51 from the crayfish monophyletic ancestors – lobsters, traveller crustaceans often reaching deep 52 depths in the ocean. These results challenge the current understanding of crayfish ecology, 53 opening an evolutionary ecological perspective which might be relevant for the next generation 54 of phylogenetical approaches. 55

To investigate crayfish respiration patterns in relation to oxygen depletion, we calculated 170 respiration rate every 30 minutes as DO decreased from 100 % saturation to anoxia (0.00 mg 171 O2/l). The Routine Metabolic Rate (RMR) was calculated every 30 minutes using the formula: 172   is the time interval (seconds) between measurements i and i+1, and M is the total wet 176 weight (g) of the crayfish used in experiment. To determine whether background respiration 177 was likely to be significant, control trials in four replicates were run within the same 178 experimental set-up, but without any crayfish in the Pârvulescu lab (Romania) and two control 179 trials were run in the Stoeckel lab (U.S.). 180 Recent studies have shown that respiration response to declining oxygen is more 181 complex and varied than traditionally recognized and the traditional, two segment, broken 182 stick model does not always fit the data well (Cobbs & Alexander, 2018). We used a four-183 segment model (TableCurve 2D v5.01; Systat Software, Inc., Richmond, CA, USA) to 184 describe respiration patterns, with regions 1-4 (R1-R4) represented by linear declines in RMR 185 alternating with stable RMR values. The breakpoints between each region were designated as 186 C1-C3. In all the cases analysed, the fits resulted in a coefficient of determination r 2 > 0.90 187 ( Fig. 1). We then calculated the mean RMR values for each region, and the DO 188 concentrations corresponding to transitions from one region to another (i.e., C1, C2, and C3). 189

190
Hypoxia and Anoxia Tolerance 191 To quantify hypoxia tolerance, we calculated lethal concentration (LC) during oxygen depletion 192 as follows: LC1 = the DO concentration at which the first crayfish died, LC50 = the DO concentration at which 50% of crayfish died, and LC100 = the DO value at which all crayfish died. Because many crayfish were still alive after DO declined to 0.00 mg/l, we also calculated 195 the average time of survival after reaching anoxia (TSARA) for each taxon. 196 197

Statistics for comparisons 198
To test whether there were any differences in mean RMR of each region (R1, R2, R3 and R4) 199 among P. leptodactylus singles and groups experiments, and to test for differences in RMR of 200 each region (i.e., C1, C2, and C3) among the three burrower categories of crayfish, we pooled 201 the RMR data from P. leptodactylus groups, and from strong, moderate, and weak burrowers, 202 run the four-segment model describing respiration patterns again, and compared among 203 burrowing categories using the non-parametric two-sample Wilcoxon test (Bauer, 1972;204 Hollander, Wolfe, & Chicken, 1973). 205 For data management, exploratory and statistical analyses, we used R 4.0.3 software 206 using the wilcox.test function. 207 208

Modelling, validation, and dynamics simulation 209
Modelling 210 In order to inspect the DO dynamics in an artificial burrow, we developed a mathematical model 211 for oxygen consumption of a virtual crayfish in a virtual burrow. A virtual crayfish with a total 212 length (TL) of 110 mm, 24 mm mean diameter (⌀) and 48 g wet weight (WW) was placed in a 213 flooded virtual cylindrical burrow 180 mm long and 38 mm diameter (⌀), connected by a 214 cylindrical tube (600, 400 and 200 mm long, 30 mm ⌀) to a cubic-shaped external tank (ET) 215 representing a part of the section of a river (or pond) in natural conditions (Fig. 2). The virtual 216 crayfish was placed with the head oriented towards the exit of the burrow. We placed the 217 consumption area (i.e., the gills) on the ventral side of the proximal half of the virtual crayfish, 218 the local convection currents generated by scaphognathites to maintain oxygen circulation were 219 simulated by imposing a local restricted velocity of 0.0001 m/s (Breithaupt, 2001; Burggren & mass flux type boundary condition (i.e., mass of DO consumed per unit time and unit surface 222 area) on the area of the active surface through which oxygen is consumed. The RMR versus 223 DO dependence was obtained from group experiments on P. leptodactylus. 224 The initial DO levels throughout the system were maintained constantly at 8.5 mg/l in 225 the ET; we simulated natural flow currents in the ET at a velocity field of 0.1 m/s, 100 mm 226 away from the entrance of the tube in the ET. The oxygen transport inside the virtual burrow 227 by convection and diffusion is described by the equation: 228 where DO is the dissolved oxygen value, t is time, v r is the velocity field, and D is the diffusion 230 coefficient of oxygen in water. 231 The walls of the burrow were considered impermeable to oxygen. The flow velocity 232 was calculated by numerically solving the classical Navier-Stokes equations for incompressible 233 fluids: 234 where  is mass density, p is pressure, and  is the dynamic viscosity of water. 237 The system of equations (3a) -(3b) was solved in the previously outlined geometry, 238 together with a non-slip condition imposed on the burrow walls, and a prescribed velocity field 239 of the water at the burrow entrance in the ET.

Experimental validation 247
To validate the mathematical model describing in-burrow DO consumption, we analysed the 248 oxygen dynamics of specimens of P. leptodactylus in a series of trials in artificial burrows 249 matching our in-silico simulation conditions. In each trial, we placed a single crayfish for 24 250 hours in a cylinder-shape plastic shelter (180 mm long, 50 mm ⌀) connected by a 38 mm ⌀ 251 cylindrical plastic tube of 200, 400 and 600 mm length to a 60 l ET filled with water at a constant 252 8.5 mg/l DO. We prevented the crayfish from escaping by placing an obstacle made of thin wire 253 threads, which did not influence the flow of water or DO variations. The oxygen sensor was 254 placed in the middle of the crayfish chamber, 15 mm from the roof, with automated recording 255 at 30-minute intervals. To mimic diffusion and convection caused by natural flow in a lotic 256 environment, water velocity of 0.1 m/s was produced in the ET using a submersible pump. 257 258

Multiple day-night cycles DO dynamics inside the burrows 259
In order to understand the DO dynamics inside crayfish burrows after multiple day-night cycles, 260 we simulated the activity of an average crayfish in a 600 mm TL, 60 mm ⌀ cylindrical burrow, 261 assuming that the crayfish was at the end of the burrow at the beginning of the simulation. The 262 variation of DO was computed assuming 12-hour cycles of activity and inactivity: 12 hours in 263 the burrow when virtual crayfish was allowed to consume the oxygen from its surroundings 264 according to previously determined RMR-DO dependence, followed by 12 hours outside the 265 burrow, a period of time when oxygen is freely redistributable in the burrow. When the crayfish 266 returned to the burrow, we assumed its location was at the most distant point from the entrance, 267 where DO is in the lower range of normoxia (DO = 6 mg/l). Preliminary trials with P. leptodactylus verified that crayfish tested together showed an 285 overall respiratory pattern similar to that of individual crayfish in terms of an initial decline 286 (RI) followed by a stable region (R2) followed by a second decline (R3) followed by a second 287 stable region (R4) regardless of sex (Fig. S1). Mean RMR was significantly different between 288 males and females only for R4 (Wilcoxon test, p=0.0277) and between singles and groups 289 mostly for R3, C1 and C3, with groups typically exhibiting a higher RMR than singles (see 290 Table 1). 291 Control runs in two different laboratories revealed average oxygen depletion rates 292 within the range of 0.05 -0.17 mg DO/l/hr in the Romanian lab and 0.09 mg DO/l/hr in each 293 of two separate runs in the U.S. lab representing a background oxygen demand of 4-9 % of 294 the uncorrected crayfish oxygen demand. Because controls were not run in all labs, we report 295 uncorrected RMR estimates only and assume true respiration rates were ≥ 90% of the reported 296 rates. 297 similar to each other than to primary burrowers. Mean respiration rate did not differ between 299 secondary and tertiary burrowers in any region (R1-R4) but was significantly higher in R2 300 and R3 for primary burrowers (Wilcoxon test p < 0.05; Table 2; Fig. 5). Similarly, none of the 301 transition points (C1-C3) differed between secondary and tertiary burrowers, but C1 occurred 302 at significantly lower DO concentrations for primary burrowers, while C3 was significantly 303 higher relative to secondary and tertiary burrowers (Wilcoxon test, p < 0.05; Table 2; Fig. 5). 304 We found no significant differences between the respiratory patterns of the analysed North 305 American and European P. clarkii specimens. 306 307

Hypoxia and Anoxia Tolerance 308
Tertiary burrowers appeared to be least tolerant of hypoxia and anoxia with two of three 309 species tested exhibiting LC50 and LC100 values > 0.00 mg/l, whereas two of three primary 310 burrowing species and all seven secondary burrowing species exhibited less than 50% 311 mortality prior to reaching anoxia (Table 3). Once anoxia was reached, two of the primary 312 burrowing species exhibited mean survival times of 13.6 and 14.5 hours respectively whereas 313 the longest mean survival times for secondary and tertiary burrowers were 9.5 and 5.7 hours 314 respectively. The aquarium species, P. virginalis, exhibited a mean anoxia survival time of 315 1.8 hours. An important exception to the anoxia tolerance of primary burrowers was P. 316 brasiliensis, which had the highest LC50 (0.24 mg/l) of any species tested and experienced 317 100% mortality before DO declined below 0.21 mg/l (Table 3). 318 319

Modelling, validation, and dynamics simulation 320
Mathematical in-silico modelling of DO availability in relation to burrow length did not differ 321 significantly from the in-vivo experiment (Fig. 3a). The simulation showed that after 30,000 s 322 (about 8 hours), all available DO might be consumed nearby the crayfish in burrows with 600-323 through convection from external water flow allowed DO to remain > 6 mg/l in some portions 326 of the burrow proximal to the crayfish (Fig. 3b). 327 Our simulations on DO consumption in a virtual burrow in multiple 12-hour day-night 328 cycles (Fig. 4) show that an immobile crayfish consumes almost all oxygen in the first 12 hours 329 when occupying burrows longer than 400 mm. In our model, the oxygen in the burrow would 330 not return to normal, pre-inhabitancy levels during the next 12 hours with no crayfish, indicating 331 that the external water-flow-induced convection is not sufficient to homogenously deliver DO 332 in the burrows with 600-and 400 mm length connecting tubes. Of note, after four such in-and-333 out cycles, these burrows were basically depleted of oxygen. Consistent with the DO modelling 334 (and in-vivo experimental measurements), the convection effect is relevant only for burrows 335 with a 200 mm connecting tube (or shorter), suggesting that only crayfish close to the ET might 336 benefit from continuous oxygen supply from outside. 337 The in-silico simulations for the air-filled burrow, specifically for primary burrowers, 338 showed a thin line of decreased oxygen only around the consumption area of the crayfish, the 339 rest of the burrow volume remaining saturated, the rate of diffusion in air conditions being much 340 higher than in water. 341 342 DISCUSSION 343

Physiological implications 344
Overall, it is expected that if forced to choose between exposure to predators in oxygenated 345 waters and the safety of hypoxic burrow waters, a wide range of crayfish species have 346 evolved physiological adaptations to withstand hypoxia or even anoxia for many hours at a 347 time. This is supported by results of this study wherein crayfish taxa from multiple continents, 348 families, and burrowing groups exhibited LC50's < 0.5 mg O2/l and were capable of surviving 349 several hours of complete anoxia. It is worth noting that the aquarium species P. virginalis behave somewhere between 448 secondary and tertiary burrowers. Changes in burrowing behaviour was reported for invasive 449 species in the new environment (Guan, 1994). Our study did not reveal significant differences 450 in respiratory behaviour between the native range versus invading P. clarkii populations. Yet, we believe these anaerobic mechanisms were preserved due to crayfish use of burrows. 487 Crayfish are susceptible to predation, cannibalism, and desiccation. Sheltering in burrows is a 488 common behaviour. Aside of ancient phylogenetic divergence, some species of crayfish (i.e., 489 parastacids), according to our results, appears unable to use anaerobic mechanisms most 490 likely due to genetic degradation for an unused heritage. Burrowing is a very old behaviour, 491 an assumption supported by the findings of crayfish-related fossils of burrows (Smith,492 Hasiotis    General RMR versus DO behaviour, revealing the respiratory predicted regimens (R1, R2, R3, R4). The stars represent the experimental values, the line describes the RMR/DO dependence obtained by the tting process, the points C1, C2, C3 represent the DO values corresponding to the RMR modelling transitions between two successive predicted respiratory regimens.

Figure 2
Schematic representation of the geometry of virtual model of cray sh burrow (the walls of the tube were considered impenetrable for oxygen) and a owing system (the cubic box in which the water is considered owing, with velocity 0.1 m/s perpendicular to the direction of the burrow). The cray sh is represented by a cylinder (detailed in the image in the left-upper corner), the purple zone representing the moving area of gills and pleopods (imposing a water current of 0,0001 m/s), and the green area represents the consumption zone (the gills).  Calculated dissolved oxygen distribution inside the burrow after successive 12-hour day and night cycles.
The model considers that the cray sh occupies the shelter and consumes oxygen during the day, while during the night, when the cray sh are supposed leaves the shelter, supplementary oxygen is provided in the burrow by diffusion from the outside water. The RMR trend comparisons for primary vs. secondary (a), secondary vs. tertiary (b) and primary vs. tertiary burrowers (c).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.