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Urine spraying and scraping as potential scent-markingThe urine spraying and the scraping were reported in other felids6,20,21. In this study, only half of the other excretion instances were accompanied by sniffing, whereas almost all urine spraying and scraping events were accompanied by sniffing, indicating that these are scent-markings. The sniffing was also often observed immediately before urine spraying and scraping. Given the significant association of sniffing before excretion, especially with regard to the scraping, the presence or absence of a scent on the object was thought to be a trigger.Furthermore, during the scraping, liquid secretions thought to originate from the anal glands, were released. Domestic cats have scent glands in the anal sac22. The presence of secretions from the anal sac has also been confirmed in not only tigers, lions (Panthera leo), and bobcats (Lynx rufus), but also in cheetahs1,6,23; however, this study was the first to investigate their role in excretion. Generally, secretions are considered to be caused by health problems or estrus, but in this study, none of the individuals had health problems, and all secretions were observed only in males. Therefore, it was thought that the secretion was produced by the scent glands and contributed to a stronger smell than only urine and feces.Variations based on sexUrine spraying was observed only in adult males and females, and was more frequent in males, as reported in other felids4,5,6,9,24. In wild cheetahs, although urine spraying and scraping have been observed as scent-making, the frequency of scent-marking is known to be substantially higher in territorial than in non-territorial males and in females15,16,25, and the marking locations are concentrated in the core area of the male territories16. The territories of a single male cheetah or a male group are relatively small and exclusive, whereas the relatively large home ranges of non-territorial males (also known as “floaters”) overlap with each other and with those of females15,16. A male’s home range is also larger than that of a female15,16,26,27. Male cheetahs rarely encounter other males because they communicate via marking posts28. Given these reports, the frequent urine spraying by males may help prevent encounters between males. In addition, observations of captive cheetahs have shown a significantly positive correlation between urinary spraying frequency and fecal estradiol content in female cheetahs19. Therefore, as Cornhill and Kerley24 mentioned, female urine spraying is caused by estrus, and male urine spraying is intended as a home range marker for other males or as a sign for females.The action of scraping using the hind paws has been reported to occur in both males and females in servals, lions, tigers, black-footed cats, etc.2,5,6,7,29; however, this behavior was only observed in adult males in this study. Sunquist and Sunquist3 reported that female cheetahs also perform the scraping. In this study, we only recorded observations when the cheetahs were released in the outdoor enclosures, and not when they were in the indoor facilities. In 43.6% of the scraping events, the males excreted feces. During the observation period, the females defecated in the indoor facilities, and no defecation was observed in the outdoor enclosures. It is possible that no scraping action was observed among the females because defecation was not observed in the outdoor enclosure. In indoor facilities, the cheetahs were in a completely monopolized enclosure; hence, the females defecated in their own spaces. There was a difference in the defecation sites and frequency of scraping between the males and females; this was attributed to the sex difference in scent-marking.Differences in target height for each behaviorUrine spraying was frequently done on objects approximately 170 cm or higher, such as walls or fences, standing trees, and stumps, whereas scraping was observed on low-lying objects on the ground, such as a straw pile approximately 3 cm high and a fallen tree that was 10–50 cm high. In other words, the cheetah engaged in urine spraying and scraping depending on the object nearby. This might indicate the functional role of these behaviors. This is consistent with previous findings of urine spraying by tigers being more frequent in wooded forests than in grasslands, with few prominent objects, and scraping being more common in the latter6. In addition, in a study that investigated the place where the smell of the urine of domestic cats is likely to remain, the smell persisted for a long time on rough surfaces, areas covered with moss, and overhanging slopes30. Even for cheetahs living in the savanna woodlands, where there are comparatively fewer upright objects than in the habitat of felids living in the forest, increasing the chances of transmitting information via not only urine spraying but also by the scraping might be more important. On the other hand, in their natural habitat, there are some large carnivores like lions and leopards (Panthera pardus). Wild cheetahs tend not to visit the sites where such carnivores’ scent-mark is present31, suggesting that they might confine their marking to specific sites devoid of other carnivores’ scent. Further research is needed to determine how wild cheetahs use urine spraying and scraping. In this study, scraping was frequently observed even on tall stumps and rocks if they were within the cheetahs’ reach. Scraping by wild cheetahs has been also observed on trees32. Zoos other than Zoo C had few prominent horizontal objects. Therefore, the presence of straw piles, fallen trees, stumps, and rocks may have elicited the scraping.Differences in housing conditionsIn zoos C and D, where animals shared enclosures, the frequency of both urine spraying and scraping by males was higher than in the males in the monopolized enclosures. They possibly showed a more frequent scent-marking to strengthen their home range claims when sharing the exhibition space15. Regarding the scraping, Zoo C had at least four low and horizontal objects (straw piles, fallen tree, stones, and rocks), and scraping was frequently observed. As mentioned above, the placement of objects might have elicited the scraping.In this study, the frequency of urine spraying decreased when the submissive individual (Male 17) was released in the enclosure where the dominant individual (Male 13) was previously released. Among wild cheetahs, territorial males have been reported to mark their territories more often than non-territorial males17,25. Therefore, the difference in the number of markings is considered to be related to whether or not the target individual is within the territory, and it is highly possible that the dominant/submissive relationship between males at that location has an effect on marking.Function of scraping using hind pawsOther felid studies have reported scraping in tigers, pumas, jaguars, clouded leopards, and small felids6,10,20,21,32,33; however, there are fewer studies on different types of scraping. In certain species, such as jaguars and pumas, scraping using hind paws is more frequent than urine spraying33. From this study, the use of secretions was confirmed in the scraping, and it was considered to be a significant marking of the cheetah.The possible functions of scraping include: (1) dispersing the smell of excrement, (2) placing the smell of excrement on the hindlegs, (3) smearing the objects with excrement, and (4) adding the scent of the hind paws. Domestic cats are known to cover their feces with soil34; however, in this study, the cheetahs did not cover the feces with soil and were not observed to scrap only after excretion. Therefore, scraping using hind paws was not meant for concealing urine and/or feces. The results of this study suggest that the scrapings were mostly performed during and after excretion for any of the aforementioned functions. However, 43.2% of the observed scraping events were performed before excretion, and in these cases, the functions 1–3 did not apply, since we did not observe the feces being crushed by scraping the hind paws. As for function 4, domestic cats have sweat glands on the soles of their feet that are thought to retain their smell35. Therefore, the sweat glands on the soles of the feet of the cheetahs possible retain the smell of the hind paws as well. Schaller36 reported that among tigers, scraping on the grassland was exhibited by scratches in the grass and exposure of the ground, creating a visual effect. In the case of cheetahs, scraping may have the function of creating grooves and ridges on the ground to enhance the visual effect; however, the formation of grooves and ridges were not observed in this study. In certain cases, they scraped against trees and stones. Because trees and stones are not easily deformed, it is hard to say whether the visual effect was enhanced by scraping with their hind paws.Scraping has been reported in other felids; however, the movement of the hindlimbs is not uniform. For example, in the case of bobcats, behaviors such as kicking back on the ground with no surrounding objects and scattering of soil have been observed during scrapings20. The snow leopard slowly moves its hindlimbs on the ground near the rocks, exposing the ground; in fact, Schaller29 observed a tiger scraping its hind paws to create a pile of soil [37; Kinoshita, personal communication: Online Resource 3; Scraping of snow leopard]. The movement of urine spraying also varies among species. For example, bobcats sometimes squat and urinate on the ground20, and snow leopards rub their cheeks against the target object and then spray urine9, but cheetahs do not rub their face before urine spraying. Hence, even in the same behavior of “spraying/scraping,” the actions differ. Because felids are widely distributed in various environments, such differences in movements are possibly related to differences in habitat and behavioral functions.In conclusion, urine spraying and scraping using hind paws were considered scent-markings because they were more strongly associated with sniffing than other excretion. Both behaviors were also observed only in adults; however, urine spraying was confirmed in both sexes and was more frequent in males than in females, whereas scraping was observed only in males. Also, the frequencies of both behaviors were significantly higher in males kept in shared enclosures containing other individuals than in males kept in monopolized enclosures, while there was no difference in the frequencies among females. Hence, there were sex differences in these scent-markings possibly because of the difference in the sociality between the sexes even in captivity; the frequency of scent-markings was affected by the living environment including the number of target objects; urine spraying was frequently done on tall objects such as walls or fences, whereas scraping was more commonly done on low-lying objects near the ground, such as straw piles. To our knowledge, this study is the first to confirm that during the scraping a liquid other than feces and urine was secreted, presumably from the anal glands. Taken together, the results can serve to enhance our knowledge regarding the behavior of cheetahs, help improve management of these animals in captivity as well as breeding and animal welfare ex situ conservation, and help elucidate the kind of habitat that should be preserved for the in situ conservation of cheetahs. More

Plants increase their freezing resistance upon exposure to low temperatureThe freezing resistance (LT50 values) was found to vary ranging from − 6.9 °C (14-August-2017) to − 31.7 °C (04-November-2018) over the course of study period. The freezing resistance of leaves recorded during the 12 sampling time-points has been provided in Table 1 (also see39). The overlap of confidence intervals around the mean was examined for comparison of LT50 values for the different sampling time-points. Significant differences in freezing resistance were observed across the sampling time-points (Table 1). Leaves of R. anthopogon collected during summer [July and August (Air temperature and photoperiod was about 9.6 °C and 13 h day−1 respectively)] showed marginal resistance to freezing (LT50: − 7 °C) and thus, are more susceptible to freezing damage. Further, as the ambient air temperature and photoperiod decreased towards the end of growing season (i.e., October and November 2017 with air temperature and photoperiod of about − 1.1 °C and 10.5 h day−1 respectively), the plants acquired the highest freezing resistance (LT50: − 30 °C). Interestingly, a sharp increase in freezing resistance (− 29.4 °C) was observed in September 2018, when the daily mean air temperature decreased below 0 °C due to sudden snowfall (Supplementary Fig. S2). Comparison of LT50 values of all the leaf samples of R. anthopogon showed that cold de-acclimation occurred after the snowmelt during early spring in June (LT50: − 13.4 °C) with an increase in air temperature and photoperiod. These results demonstrated that R. anthopogon plants exhibit lowered freezing resistance during the warmer months [hence, these time-periods were referred as non-acclimation (NA)], progressively develop greater freezing resistance during the onset of winter season (hence, referred as cold acclimation) followed by an intermediate level of freezing resistance during the spring [hence, these time-periods were referred as de-acclimation (DA)].Table 1 The estimates of LT50, calculated by fitting sigmoidal curve to electrolyte leakage values of temperature treatments, recorded for leaves collected during the different sampling time-points (from August 22, 2017 to September 18, 2018).Full size tableDuring the acclimation period (i.e., late in the growing season), plants acquired the highest resistance to freezing (Fig. 1). The low electrolyte leakage (= high freezing resistance) observed during this period might be due to changes in cell wall properties (such as increase in lignification and suberization of cell walls), which provide resistance to diffusion of electrolytes from cells of the leaves to the extracellular water47. Moreover, high freezing resistance may also be attributed to high leaf toughness and sclerophyllous habit of this evergreen species48. Further, it was found that freezing resistance was the lowest during mid-summer period. This pattern could be explained by a trade-of between plant growth rates and freezing resistance, where warmer temperatures favour plant allocation to growth49. These observations corroborated well with earlier reports that showed a rapid increase in ‘freezing resistance’ during the transition from summer to early winter and vice versa50.Figure 1LT50 [black point (with solid fill) on the curve] calculated by fitting sigmoidal curve to relative electrolyte leakage (REL %) values recorded during the three different acclimation phases. GOF indicates ‘goodness of fit’ test values for the fitted sigmoidal curves.Full size imagePhotosynthetic rates are higher during non-acclimation and de-acclimation periodIt was found that PN of R. anthopogon varied in the range from 8.336 to 17.64 μmol(CO2)m−2 s−1 and E from 2.281 to 4.912 mol(H2O)m−2 s−1, throughout its growing season. The Gs of leaves was estimated to be in the range from 0.110 to 0.265 mol (H2O) m−2 s−1. WUE, a ratio of PN and E, varied between 52.21 and 87.68 (Table 2). The gas exchange parameters of R. anthopogon varied significantly among the sampling time-points [referred to here as different acclimation phases of the growing period of evergreen shrub (Fig. 2, Table 3)]. In particular, PN was significantly lower on 18-September-2018 (referred as cold acclimation phase), whereas it was higher on 31-August-2018 and 15-June-2018 (referred as NA and DA phases, respectively). Similarly, Gs of leaves was significantly lower during cold acclimation in comparison to the rest of the acclimation phases (i.e., NA and DA). Further, WUE was significantly higher during cold acclimation, while it was lower during both NA and DA (p ≤ 0.05) (Fig. 2).Table 2 Variability in leaf gas exchange parameters of R. anthopogon during the different acclimation phases (NA = Non-acclimation, LA = Late cold acclimation and DA = De-acclimation).Full size tableFigure 2Variability in leaf gas exchange parameters of R. anthopogon during the three acclimation phases [i.e., Non-acclimation (31 August, 2018), Cold acclimation (18 September, 2018) and De-acclimation (15 June, 2018)]. Different alphabets (a, b, c) represent statistically significant values (p  More

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Overall strategyThe aim of this project was to synthesize publicly available data on land use, pesticide use, and toxicity to generate a ‘toolkit’ of data resources enabling improved landscape-scale research on pesticide-pollinator interactions. The main outcomes are several novel datasets covering ten major crops or crop groups in each of the 48 contiguous U.S. states:

I)

Average application rate (kg/ha/yr) of >500 common pesticide active ingredients (1997–2017),

II)

Aggregate bee toxic load (honey bee lethal doses/ha/yr) of all insecticides combined (1997–2014), (Note that this dataset ends in 2014 because after that year, data on seed-applied pesticides were excluded29, and these contribute significantly to bee toxic load21)

III)

Reclass tables relating these pesticide-use indicators to land use/land cover classes to enable the creation of maps predicting annual pesticide loading at 30–56 m resolution.

An overview of the steps, inputs, and outcomes are provided in Fig. 1.Fig. 1Overview of the data synthesis workflow described in this paper.Full size imageData inputsA summary of input datasets is provided in Table 1.Table 1 Data inputs used in this study.Full size tablePesticide dataPesticide use data were last downloaded from the USGS National Pesticide Synthesis Project30,31 in June 2020. This dataset reports total kg applied of 508 common pesticide active ingredients by combinations of state, crop group, and year for the contiguous U.S. from 1992–2017 (crop groups explained in Table 2). The data are derived primarily from farmer surveys conducted by a private firm (Kynetec). For California, USGS obtains data from the state’s pesticide use reporting program32. USGS then aggregates and standardizes both data sources into a common national dataset that is released to the public and was used in this effort. The USGS dataset includes both a ‘high’ and a ‘low’ estimate of pesticide use, varying based on the treatment of missing values in the source data31. Because previous work on this dataset suggested that the ‘low’ estimate more closely matches independent pesticide estimates33, we used the ‘low’ estimate throughout, but assess the influence of this choice on the resulting estimates (see Technical Validation). While we focus on the ‘low’ estimate for the data and outputs presented in this manuscript, the workflow we developed can accommodate both the low and high estimates.Table 2 USGS crop categories in pesticide source data, based on metadata from USGS30,31 and personal communication with USGS staff scientists.Full size tableCrop area dataTo translate pesticide use estimates into average application rates, it was necessary to divide total kg of pesticide applied by the land area to which it was potentially applied. Crop area data were last downloaded from the Quick Stats Database of the USDA34 in May 2020, using data files downloaded from the ‘developer’ page. This USDA dataset contains crop acreage estimates generated from two sources: the Census of Agriculture (Census), which is comprehensive but conducted only once every five years35 and the crop survey conducted by the National Agricultural Statistics Service (NASS), which is an annual survey based on a representative sample of farmers in major production regions for a more limited subset of crops36.Honey bee toxicity dataTranslating insecticide application rates into estimates of bee toxic load (honey bee lethal doses/ha/yr) required toxicity values for each insecticide active ingredient in the USGS dataset. We used LD50 values for the honey bee (Apis mellifera) because this is the standard terrestrial insect species used in regulatory procedures, and so has the most comprehensive data available. This species is also of particular concern as an important provider of pollination services to agriculture. As previously reported21, the LD50 values were derived from two sources, the ECOTOX database37 of the U.S. Environmental Protection Agency (US-EPA), and the Pesticide Properties Database (PPDB)038. ECOTOX was queried in July 2017, by searching for all LD50 values for the honey bee (Apis mellifera) that were generated under laboratory conditions. Acute contact and oral LD50 values for the honey bee were recorded manually from the PPDB in June 2018.Land cover dataMapping pesticides to the landscape requires land use/land cover data indicating where crops are grown. We used the USDA Cropland Data Layer (CDL)39, a land cover dataset at 30–56 m resolution produced through remote sensing. This dataset is available starting in 2008 for states in the contiguous U.S., with some states (primarily in the Midwest and Mid-South) available back to the early 2000s.Data preparationRelating datasetsA major challenge in this data synthesis effort was relating the various data sources to each other, given that each dataset has unique nomenclature and organization. We created the following keys (summarized in Table 3) to facilitate joining datasets:

I)

USGS-USDA crop keys – Using documentation and metadata associated with the USGS pesticide dataset31,33,40, we created keys relating the USGS surveyed crop names (‘ePest’ crops) and the ten USGS crop categories to the large number of corresponding crop acreage data items in the Census and NASS datasets. For annual crops and hay crops we used ‘harvested acres,’ and for tree crops we used ‘acres bearing & non-bearing.’ These choices were made to maximize data availability and to correspond as closely as possible to the crop acreage from which the pesticide data were derived31. A separate key was developed for California because California pesticide data derives from different source data and covers a larger range of crops.

II)

USGS-CASRN compound key – Using USGS documentation as well as background information on pesticide active ingredients38,41, we generated keys relating USGS active ingredient names to chemical abstracts service (CAS) registry numbers to facilitate matching compounds to the ECOTOX and PPDB databases.

III)

USGS compound-category key – In this key we classified active ingredients into major groups (insecticides, fungicides, nematicides, etc.) and into mode-of-action classes on the basis of information from pesticide databases and resistance action committees38,41,42,43,44.

IV)

USGS-USDA compound key – To facilitate our data validation effort, we generated a key relating USGS compound names to USDA compound names, on the basis of information from several pesticide databases38,41.

V)

USGS-CDL land use-land cover keys – Using documentation from the USGS pesticide dataset describing the crop composition of each of the ten crop categories31, we created a key that matches these categories to land cover classes in the CDL. A separate key was developed for California given the differences in surveyed crops in this state, noted above.

Table 3 Keys generated to relate datasets.Full size tableProcessing crop area dataBecause of differences in the crops included in pesticide use estimates, crop acreage data were processed separately for California and for all other states, and then re-joined, as follows: Acreage data were first filtered to include only data at the state level, reporting total annual acreage for states in the contiguous U.S. after 1996. Acreage data were joined to the appropriate USGS-USDA crop key and only those crops represented in the pesticide dataset were retained. We then generated an acreage dataset with single rows for each combination of crop, state, and year using data from the Census when available (1997, 2002, 2007, 2012, 2017), data from NASS in non-Census years, and temporal interpolation to fill in remaining missing values (i.e. linear interpolation between values in the same state and crop in the nearest surrounding years). This process was repeated for California, using acreage data for only that state in combination with the CA crop key. Finally, acreage data in the two datasets were recombined, converted to hectares, and summed by USGS crop group.Processing honey bee toxicity dataProcessing for the honey bee toxicity data has been described in detail elsewhere21. Briefly, toxicity values were categorized as contact, oral, or other and standardized where possible into µg/bee. Records were retained if they represented acute exposure (4 days or less) for adult bees representing contact or oral LD50 values in µg/bee. To generate a consensus list of contact and oral LD50 values for all insecticides reported in the USGS dataset, we gave preference to point estimates and estimates generated through U.S. or E.U. regulatory procedures, taking a geometric mean if multiple such estimates were available. Unbounded estimates (“greater than” or “less than” some value) were only used when point estimates were unavailable, using the minimum (for “less than”) or the maximum (for “greater than”). If values for a compound were unavailable in both datasets, we used the median toxicity value for the insecticide mode-of-action group. And finally, in rare cases (n = 1/148 compounds for contact toxicity and 8/148 compounds for oral toxicity) we were still left without a toxicity estimate for a particular insecticide. In those cases, we used the median value for all insecticides.Data synthesisCompound-specific application rates for state-crop-year combinationsUSGS data on pesticide application were joined to data on crop area. Average pesticide application rates were calculated by dividing kg applied by crop area (ha) for each combination of compound, crop group, state, and year.Aggregate insecticide application rates for state-crop-year combinationsThe dataset from the previous step was filtered to include only insecticides, and then joined to LD50 data by compound name. Bee toxic load associated with each insecticide active ingredient was calculated by dividing the application rate by the contact or oral LD50 value (µg/bee) to generate a number of lethal doses applied per unit area. These values were then summed across compounds to generate estimates of kg and bee toxic load per ha for combinations of crop group, state, and year.Missing values were estimated using temporal interpolation, where possible (i.e. linear interpolation between values in the same state and crop group in the nearest surrounding years). This dataset ends in 2014 because after that year seed-applied pesticides were excluded from the source data29, and they constitute a major contribution to bee toxic load21.We focused bee toxic load on insecticides for three reasons. First, quality of LD50 data is highest for insecticides and uneven for fungicides and herbicides. Point estimates make up the majority of LD50 values for insecticides, whereas  100 µg/bee”, increasing the uncertainty of downstream estimates). Second, insecticides tend to have greater acute toxicity toward insects than fungicides and herbicides (median [IQR] LD50 = 100 [44–129] µg/bee for fungicides, 100 [75–112] µg/bee for herbicides, and 1.36 [0.16–12] µg/bee for insecticides). As a result, insecticides account for > 95% of bee toxic load nationally, even when herbicides and fungicides are included (and even though insecticides make up only 6.5% of pesticides applied on a weight basis). Third, focusing these values on insecticides increases their interpretability, reflecting efforts directed toward insect pest management, rather than a mix of insect, weed, and fungal pest management (which often have distinct dynamics and constraints for farmers).While we chose to include only insecticides in this aggregate value, users are welcome to adjust the workflow to include fungicides and herbicides if desired. To this end, we provide our best estimates for LD50 values for fungicides and herbicides in the USGS dataset (Table 4).Table 4 Data outputs generated by this study.Full size tableReclassification tablesTo generate reclassification tables for the CDL, the pesticide datasets described above were joined by crop group to CDL land use categories. The output of these processes was a set of reclassification tables for combinations of compound, state, and year. Also generated was a set of reclassification tables for aggregate insecticide use for combinations of state and year.Of the 131 land use categories in the CDL, 16 represent two crops grown sequentially in the same year (double crops, found on ~2% of U.S. cropland in 201245), which required a modified accounting in our workflow. Pesticide use practices on double crops are not well described, but one study suggested that pesticide expenditures on soybean grown after wheat were similar to pesticide expenditures in soybean grown alone46. Therefore, we assumed that pesticide use on double crops would be additive (e.g. for a wheat-soybean double crop, the annual pesticide use estimate was generated by summing pesticide use associated with wheat and soybean).Missing values in the reclassification tables resulted from several distinct issues. Some values were missing because a particular crop was not included in the underlying pesticide use survey (e.g. oats was not included in the Kynetec survey), or because the land use category was not a crop at all (e.g. deciduous forest). These two issues were indicated with values of ‘1’ in columns called ‘unsurveyed’ and ‘noncrop,’ respectively. For double crops, a value of 0.5 in the ‘unsurveyed’ column indicates that one of the crops was surveyed and the other was not. For compound-specific datasets, missing values may reflect that a given compound was not used in a state-crop group-year combination. For the aggregate insecticide dataset, even after interpolation there were some missing values, usually when a state had very little area of a particular crop or crop group.Finally, missing data for double crops were treated slightly differently in the aggregate vs. compound-specific reclassification tables. For the aggregate insecticide dataset, estimates for double crops were only included if estimates were available for both crops; otherwise the value was reported as missing. For the compound-specific datasets, estimates for double crops were included if there was an estimate for at least one of the crops, since specific compounds may be used in one crop but not another. More

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