{'fraction_non_alphanumeric': 0.02355842384175455, 'fraction_numerical': 0.03767249068681463, 'mean_word_length': 4.82518337408313, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 1, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Phenotypic plasticity, but not genetic adaptation, underlies seasonal variation in the cold hardening response of Drosophila melanogaster

In temperate regions, an organism’s ability to rapidly adapt to seasonally varying environments is essential for its survival. In response to seasonal changes in selection pressure caused by variation in temperature, humidity, and food availability, some organisms exhibit plastic changes in phenotype. In other cases, seasonal variation in selection pressure can rapidly increase the frequency of genotypes that offer survival or reproductive advantages under the current conditions. Little is known about the relative influences of plastic and genetic changes in short lived organisms experiencing seasonal environmental fluctuations. Cold hardening is a seasonally relevant plastic response in which exposure to cool, but nonlethal, temperatures significantly increases the organism’s ability to later survive at freezing temperatures. In the present study, we demonstrate seasonal variation in cold hardening in Drosophila melanogaster and test the extent to which plasticity and adaptive tracking underlie that seasonal variation. We measured the cold hardening response of flies from outdoor mesocosms over the summer, fall, and winter. We bred outdoor mesocosm-caught flies for two generations in the lab and matched each outdoor cohort to an indoor control cohort of similar genetic background. We measured the cold hardening response of indoor and field-caught flies and their laboratory-reared F1 and F2 progeny to determine the roles of seasonal environmental plasticity, parental effects, and genetic changes on cold hardening. We also tested the relationship between cold hardening and other factors, including age, developmental density, food substrate, presence of antimicrobials, and supplementation with live yeast. We found strong plastic responses to a variety of field- and lab-based environmental effects, but no evidence of seasonally varying parental or genetic effects on cold hardening. We therefore conclude that seasonal variation in the cold hardening response results from environmental influences and not genetic changes.


Introduction
with an increase in cold tolerance resulting from pre-exposure periods as brief as half an hour 73 (Czajka and Lee 1990). Cold hardening has been documented in larvae, pupae, and adults. conditions. In addition to genetic influences, specific aspects of environmental exposure, such as 85 the temperature and duration of cold exposure (Cjazka and Lee 1990; Kelty and Lee 1999) also 86 affect cold hardening. We thus hypothesized that the cold hardening response might vary 87 seasonally in temperate climates via some combination of genetic adaptation and environmental 88 influences. 89 In the present study, we ask whether the cold hardening response in D. melanogaster 90 varies seasonally in field-reared populations, and if so, whether this variation occurs as a result 91 of environmental influences, genetic changes, or some combination thereof. Although other 92 studies have examined the occurrence of rapid cold hardening in the field (e.g. Kelty 2007; 93 Overgaard and Sorensen 2008), in this study we measure the effect of seasonal changes on the 94 response to a consistent cold hardening treatment. Over the course of multiple seasons in a single 95 year, we collected flies from outdoor mesocosms and then subjected them to a controlled pre-96 exposure to cool temperatures followed by a freeze tolerance test. By contrasting outdoor-caught 97 flies, their lab-reared offspring, and their grandchildren to similarly treated flies that were reared 98 entirely indoors, we found that cold hardening does not evolve over seasonal timescales but 99 shows dramatic season-specific plasticity. We further show that this plasticity is potentially 100 caused by thermal exposure in the field and is likely influenced by nutrition, age, and other 101 factors. Taken together, our work suggests that cold hardening is a highly plastic trait that does  139 Temperature data recorded during a representative freeze assay. Temperature probes exposed to 140 air inside the freezer recorded temperature fluctuations ranging from approximately -3.3 °C to -141 5.7 °C over the course of the freeze assay. Temperature probes submerged in a saltwater bath 142 inside the freezer recorded temperature fluctuations ranging from approximately -5.1 °C to -5.4 143 °C.

144
Following precooling, we noted the number of flies that had died during the precooling 145 process prior to conducting the freeze assay. We transferred each vial of flies to a 5 mL snap-cap 146 Falcon tube and suspended the tubes in the salt solution held at approximately -5 °C within a 147 chest freezer. We used weighted blocks to keep the tubes submerged in liquid up to the rim of 148 the cap. In order to minimize temperature fluctuations in the water bath, we added tubes into the 149 bath in small groups for each time point and removed all the tubes at the end of the assay. At the 150 conclusion of the freeze, we transferred flies into their original vials containing food and held the 151 food vials upside down so that unconscious flies would not become stuck in the food. The next 152 day, we recorded the number of survivors or the number dead within each vial (whichever 153 number was smaller). Flies that exhibited the ability to stand stably and walk were considered to 154 be alive, while flies that were immobile, or flies that exhibited spastic motions such as twitching 155 but were not stable in their movements and stance, were considered dead following similar 156 definitions of Czajka and Lee (1990) and Gerken et al (2015). each added to the cages weekly until November, when feeding was done biweekly) and were 163 initially inoculated with live yeast. Cages 7-11 were fed weekly with 2.5 L of cornmeal-molasses 164 fly food, the same food that fed the indoor cages. We collected "F0" flies from the outdoor 165 cages using nets or aspirators. Note that although these flies are actually advanced generations of 166 the outbred populations, we refer to them as F0s for the given collection point. On the same day, 167 we collected F0 flies from the indoor cages. We subjected males from the indoor and outdoor 168 cages to the precooling and freeze assays as described above. We used only males because  In the lab, we established 25 isofemale lines from each cage in vials. We screened the 174 offspring of the isofemale lines for presence of D. simulans and discarded these lines (on 175 average, fewer than 10/150 lines per collection time point). We combined all D. melanogaster 176 F1s together to collect males for the freeze assay and to establish F2s. F1 males collected from 177 the isofemale lines were precooled, frozen, and assayed for survival. The F1 flies were 178 propagated in bottles and the F2 males were precooled, frozen, and assayed for survival. Heavy 179 rain limited the 7/24/18 collection; we obtained sufficient F0 females to establish lines but not 180 enough males to generate a freeze survival curve. All F1 isofemale lines from the 11/7/18 and 181 11/21/18 collections were lost in an incubator failure, so we were unable to assay the offspring of 182 these collections. We collected again on 11/30/18 to replace these samples but did not perform a 183 freeze assay on the 11/30/18 F0s. 184 We downloaded weather data from a station located at Carter Mountain, which is 185 approximately 2 km from our field site, to obtain information on average daily temperatures 186 during our experiments (https://www.wunderground.com/dashboard/pws/KVACHARL73).

187
Nutrition and antimicrobial assays. We made standard cornmeal-molasses fly food in a 188 small batch using a hot plate and added standard amounts of Tegosept and propionic acid to half 189 the batch (Table 1). We used Agricor Fine Yellow Cornmeal and Golden Barrel Blackstrap containing standard cornmeal-molasses fly food, precooled them, and subjected them to the and continued to supplement these bottles with yeast throughout the development of the flies. 201 We collected adult males from each treatment and placed them in vials with standard fly food 202 and no live yeast. We precooled them for two weeks and subjected them to the freeze assay.

203
Age assay. We collected embryos from the indoor hybrid swarm cages at two-week 204 intervals for six weeks and reared them to adulthood. We passaged the adult flies to fresh food 205 weekly to prevent eclosion of new adults. We collected adult males once the youngest of the 206 cohorts had eclosed, and we then precooled, froze, and measured survival of flies from all three 207 age cohorts in a single assay.

208
Density assay. We based our density assay on a previous study (Henry et al 2018). We 209 used four density levels: approximately 5 embryos/mL, 40 embryos/mL, 120 embryos/mL, and 210 300 embryos/mL of fly food. We collected embryos from the indoor cages on cornmeal-molasses 211 fly food plates and counted embryos into vials containing 2 mL of cornmeal-molasses fly food.

212
As higher density vials took longer to develop, we waited to collect adults until every vial had

225
Effects of seasonal exposure on cold hardening. 226 To test the hypothesis that seasonal conditions influence cold hardening, we collected 227 monthly samples of outbred flies reared in fruit-fed outdoor mesocosms and cornmeal-molasses-228 fed laboratory cages and assessed their cold tolerance following two weeks of precooling in the 229 lab. We used the resulting survival curves to calculate the time required to kill 50% of the flies 230 (hereafter, "LT50"). From June until early November, the LT50 of fruit-fed, outdoor F0 flies was 231 significantly lower than the LT50 of indoor F0 flies (Figure 2A; Table 2). However, in late 232 November, the outdoor F0 flies had an LT50 greater than that of the indoor F0 flies (Figure 2A;   Table 2). However, in 244 the late November collection, we observed an increased LT50 for outdoor flies compared to 245 indoor flies (P = 5.31 x 10 -7 ). B&C) The F1 and F2 generations were reared in laboratory 246 conditions. We generally did not observe differences between LT50 values for outdoor and 247 indoor flies in the F1 and F2 generations, regardless of collection time ( Table 2). Error bars 248 represent standard error of the LT50. Standard error for F0 indoor data from November 7 th , 2018 249 is set to zero for clarity (SE = 2927.8 due to incomplete survival curve).
In the field experiment, we observed that the effects of the environment prior to 251 precooling persisted through the two-week precooling period. In order to test whether the 252 thermal environment experienced by the flies before precooling influenced their cold hardening, 253 we tested for a relationship between the average temperature on the day of collection and the 254 difference in the cold hardening response of outdoor and indoor F0 flies. We observed a 255 significant negative correlation between the average temperature on the day of collection and the  To test for transgenerational effects of seasonal exposure on the cold hardening response, 268 we compared the lab-reared F1 offspring of flies collected indoors to the lab-reared F1 offspring 269 of flies collected outdoors in a common garden assay. We observed that the cold hardening 270 response was generally consistent between the lab-reared offspring of flies collected from indoor 271 and outdoor cages ( Figure 2B, Table 2). The similarity between indoor and outdoor F1 flies 272 suggests that the differences in the cold hardening responses in the F0 flies were not passed on to 273 their offspring. We note that although indoor and outdoor LT50s were significantly different for the 6/26/18 collection (Table 2), we did not observe a consistent difference or pattern in indoor 275 versus outdoor F1 cold hardening responses. 276 We tested for genetic changes in the cold hardening response by examining the lab-reared 277 F2 offspring of flies from indoor and outdoor cages. As in the F1s, we also observed little 278 difference in the cold hardening response between outdoor and indoor flies ( Figure 2C; Table   279 2). We note that the difference in LT50 was significant for the 9/18/18 collection, but again we 280 did not observe a consistent difference or pattern between the indoor and outdoor F2 cold 281 hardening responses. Therefore, our data do not provide evidence of seasonal genetic changes in 282 cold hardening. Although we observed some differences in the overall cold hardened freeze 288 Surprisingly, we observed that F2 flies were more freeze tolerant following cold 289 hardening than their F1 parents for some collections ( Figure 2B-C, compare July F1s and F2s). bottles. We investigated whether container type was a potential cause by rearing the 9/18/18 set 293 of F1 flies in both bottles and vials. We did not observe significant differences in the cold 294 hardening response resulting from differences in container type (Table 3; P = 0.191), though 295 vial-reared flies had a slight increase in the cold hardening response compared to bottle-reared 296 flies. We suggest that differences in freeze tolerance between the F1 and F2 generations may   Table 3; P = 1.78 x 10 -9 ). We also tested whether adding antimicrobials influenced cold 331 hardening, since the cornmeal-molasses food contained Tegosept and propionic acid while the 332 rotting fruit did not. We did not observe an effect of antimicrobial presence on cold hardening ( Figure 5A, Table 3; P = 0.95). Therefore, we suggest the cornmeal-molasses diet improved the 334 indoor F0 cold hardening response relative to outdoor flies during summer months (Figure 2A) 335 and also improved the cold hardening response of outdoor flies fed cornmeal-molasses food 336 (Figure 4).  We also hypothesized that yeast availability could contribute to differences in cold  Table 3; P = 1.52 x 10 -8 ). Although differences in age structure could potentially explain the 369 lower cold hardening exhibited by fruit-fed outdoor F0 flies as compared to indoor F0 flies, we 370 expect that age structure between the fruit-fed and cornmeal-molasses-fed outdoor cages (shown 371 in Figure 4) should be similar. If age structure alone was causing the outdoor F0 flies to have a 372 lower cold hardening response in the summer and fall, we would expect the cornmeal-molasses-373 fed outdoor cages to also have decreased cold hardening, which was not the case.

374
Density is a final possibly causal factor in the indoor-outdoor cold hardening differences, 375 since different food substrates and different age structures could lead to different larval densities. 376 We reared larvae at varying densities and observed an increase in cold hardening ability at 377 relatively high densities only ( Figure 5D; Table 3; P = 0.0063). We suggest that differences in 378 density may have contributed to the observed cold hardening differences in the outdoor and 379 indoor cages; however, factors such as thermal environment and nutrition are more compelling 380 causal factors given our experimental results. temperate environments. Some organisms exhibit phenotypic plasticity, meaning that an 384 environmental stimulus induces a change in the phenotype. Some populations adaptively track, 385 meaning that some genotypes are more favorable in a given season, and therefore, individuals 386 having those genotypes will be more abundant during that season. In our study, we asked 387 whether the cold hardening response in D. melanogaster varies seasonally and whether such 388 variation is a product of plasticity or adaptive tracking. We found that the cold hardening 389 response increases as outdoor temperature decreases at the onset of winter. We also determined 390 that, while cold hardening is highly plastic, the trait does not undergo seasonal evolution. hardening treatment. In our work, we further exposed flies to a consistent precooling treatment 398 after they were exposed to field conditions. This experimental design allowed us to elucidate the 399 effects of field exposure that persisted through a consistent, controlled cold hardening regime. 400 We found that the onset of winter conditions correlated with an increased cold hardening 401 response for outdoor flies (Figures 2A, 3). Previous data have shown that the cold tolerance of  The cold hardening response does not evolve over seasons.
hardening response (Figure 2B&C; Table 2). One possible explanation is that our outbred 421 populations of D. melanogaster carried limited heritable variation in cold hardening. However, 422 two studies have demonstrated heritable variation in this trait in flies from North Carolina, a 423 population that was included in our hybrid swarms (Gerken et al 2015, 2018). Thus, we suggest 424 that the absence of adaptive tracking in cold hardening is not due to a lack of genetic variation 425 for this trait within the experimental population.  The cold hardening response varies seasonally; however, we did not observe evidence of 453 adaptive tracking. Combined with correlations between the cold hardening response and outdoor 454 temperature (Figure 3), we conclude that the seasonal variation observed was a result of 455 plasticity. However, the thermal environment is likely not the sole stimulus that triggers plastic 456 changes in the cold hardening response. 458 We demonstrated that the cold hardening response of flies reared on banana-based food is 459 significantly less than the cold hardening response of flies reared on cornmeal-molasses food 460 ( Figure 5A; Table 3). The cornmeal-molasses food contained slightly more fat and more than 461 double the sugar of the banana food (Table 1)

493
Taken together, we suggest that feeding flies increased yeast may prompt them to prioritize 494 reproduction over survival, thereby reducing energetic investment in processes related to cold hardening. If the fruit-fed cages had high levels of yeast growth in the summer that diminished in 496 the late fall, these changes could have contributed to seasonal variation in cold hardening. Our 497 observation that dietary yeast influences the cold hardening response is further evidence that cold 498 hardening is a plastic phenotype that responds to nutritional conditions.

499
Effects of life history traits on the cold hardening response. 500 The outdoor and indoor cages likely varied in density and age structure, and these factors 501 could plausibly contribute to the observed differences in the cold hardening response. We found 502 that the cold hardening response declines with age in lab-reared flies, perhaps suggesting an age-  The outdoor cages contained a large volume of fruit, perhaps resulting in lower larval 514 densities relative to the indoor controls. We found that high developmental density results in an 515 increased cold hardening response ( Figure 5D). Previous work has shown that high larval