An Observational Study of Ballooning in Large Spiders: Nanoscale Multi-Fibers Enable Large Spiders’ Soaring Flight

The physical mechanism of aerial dispersal of spiders, “ballooning behavior,” is still unclear because of the lack of serious scientific observations and experiments. Therefore, as a first step in clarifying the phenomenon, we studied the ballooning behavior of relatively large spiders (heavier than 5 mg) in nature. Additional wind tunnel tests to identify ballooning silks were implemented in the laboratory. From our observation, it seems obvious that spiders actively evaluate the condition of the wind with their front leg (leg I) and wait for the preferable wind condition for their ballooning takeoff. In the wind tunnel tests, as yet unknown physical properties of ballooning fibers (length, thickness and number of fibers) were identified. Large spiders, 16–20 mg Xysticus species, spun 50 to 60 nanoscale fibers, with a diameter of 121 to 323 nm. The length of these threads was 3.22 ± 1.31 m (N = 22). These physical properties of ballooning fibers can explain the ballooning of large spiders with relatively light updrafts, 0.1–0.5 m s-1, which exist in a light breeze of 1.5–3.3 m s-1. Additionally, in line with previous research on turbulence in atmospheric boundary layers and from our wind measurements, it is hypothesized that spiders use the ascending air current for their aerial dispersal, the “ejection” regime, which is induced by hairpin vortices in the atmospheric boundary layer turbulence. This regime is highly correlated with lower wind speeds. This coincides well with the fact that spiders usually balloon when the wind speed is lower than 3 m s-1.


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Some spiders from different families, such as Linyphiidae (sheet-weaver spiders), Araneidae (orb-weaving 32 spiders), Lycosidae (wolf spiders) and Thomisidae (crab spiders) can disperse aerially with the help of their silks, 33 which is usually called ballooning behavior [1][2][3][4][5][6]. There are two representative takeoff methods in ballooning 34 flight; "tiptoe" and "rafting" [7-10]. If spiders perceive appropriate weather conditions for ballooning, they climb 35 up to the highest position of a blade of grass or a branch of a tree and raise their abdomen as if standing on their 36 tiptoes, in order to position the abdomen at the highest level, before spinning the ballooning lines. They release a 37 single or a number of silks in the wind current and wait until a sufficient updraft draws their body up in the air. 38 This is known as a "tiptoe" takeoff [9,10] (see S1, S2). Another takeoff method is called "rafting," where spiders 39 release the ballooning lines from a hanging position relying on their drag line [7,8,10] (see S3). In this way, some 40 spiders can travel passively hundreds of kilometers and can reach as high as 4.5 kilometers above sea level [11,12]. 41 For example, one of the first immigrant species on new-born volcanic islands are known to be spiders [13][14][15]. 42 Aerial dispersal of spiders is an influential factor on agricultural economy and ecology, because spiders are highly 43 ranked predators in arthropods and impact on a prey's population [16]. Due to the spider's incredible aerial 44 dispersal ability, the physical mechanism of a spider's flight has been questioned for a long time, not only in public 45 media but also in scientific research [16][17][18][19][20][21][22][23]. 46 Ballooning dispersal is efficiently used by spiderlings (young spiders) just a few days after eclosion from their 47 eggs) to avoid cannibalism at their birth sites, which are densely populated by hundreds of young spiders, and to 48 reduce competition for resources [23,24]. Some adult female spiders balloon to find a place for a new colony 49 [4,25,26] and others balloon to search for food and mates [4,27]. Most of the ballooning spiders were spiderlings 50 and spiders under 3 mm in length and 0.2 to 2 mg in mass [1][2][3][4][5]28,29]. Nevertheless, there are only a few reports 51 on the ballooning of large spiders (over 3 mm in length, over 5 mg in mass) [4,5,25,26]. 52 Spiders balloon most frequently during late spring and autumn seasons [2,4,30]. The influences of microclimates 53 on ballooning, such as temperature, humidity and wind conditions, have been extensively studied: (i) Many studies 54 agree on a positive correlation of temperature [1,3,31] or a rapid increase in temperature [31-34]; (ii) low humidity 55 is favorable for spiders to balloon [1,32,34]; (iii) for small spiders, 0.2-2 mm in length, the favorable mean wind 56 speed is limited to 3 m s -1 at a level of 2 m [30,31,35]. The local favorable wind speeds were 0.35-1.7 m s -1 in 57 experiments and 0.55-0.75 m s -1 in nature [1,3]. These values, however, differ for spiders of different sizes 58 (between 0.78-1.21 mm) [1,36]. Recently, Lee et al. showed that not only the mean wind speed at a level of 2 m 59 but also the local wind speed can be limited by a wind speed of 3 m s -1 for spiderlings [37]. Instability of of spiders' ballooning behavior with atmospheric turbulent flow [16,20]. 62 There have been a number of models that have tried to explain spiders' high buoyant capability (aerial dispersal 63 capability): a fluid-dynamic lollipop model [17], a flexible filament model in turbulence [16] and an electrostatic 64 flight model [22]. Recently, Zhao et al. implemented the two-dimensional numerical simulation using an immersed 65 boundary method, which can simulate the ballooning dynamics in more detail [38]. The result shows that the 66 atmospheric instability enables longer suspension of a ballooner in the air, which agrees with the result of 67 Reynolds's simulation and suggests that a spider may sense the vibration of vortex shedding on the spider silk 68 through their silk [16,38], which is an interesting hypothesis. 69 In spite of the abovementioned models and studies, dynamics in spiders ballooning are still not well understood, 70 because of a lack of serious scientific observation studies and specific experiments. Many of the ballooning spiders 71 are very small with weights of 0.2-2 mg, which are difficult to study [1,3,36,37]. Many described experiments 72 were not focusing on the spider's ballooning behavior itself, but assumed that spiders use a certain length of the 73 drag line [18,19,21]. The ballooning of large spiders is also a struggle because of (i) the observed physical 74 properties of ballooning silks and spider size (60-80 cm long and 3-4 silk threads, 85 to 150 mg body weight) of 75 an adult of Stegodyphus mimosarum seemed to be unrealistic for ballooning [25], because the required vertical 76 The aim of this paper is to offer behavioral clues and quantitative data in ballooning flight that may answer these 84 questions. Therefore, we investigated the ballooning behavior of adult and subadult crab spiders (Xysticus species, 85 Thomisidae), that had a size of 3-6 mm and a weight of 6-25 mg. This observation of large spiders could provide 86 a good basis for the physical characterization of ballooning. Additional experiments were performed in a wind 87 tunnel, for a precise documentation of ballooning silks and to analyze the details of ballooning behavior. Also, the 88 aerodynamic environment on a flat grass field was measured to investigate the usable updraft for a ballooning Twelve crab spiders (9 females and 3 males, adult or subadult) were used for the wind tunnel experiment. Pre-135 ballooning behavior of these spiders was induced in front of an open jet wind tunnel in which the diameter of the 136 nozzle exit is 0.6 m (see Fig. 1A). The spinning behaviors that led to the ballooning silks was observed precisely. 137 There were no obstacles next to the wind tunnel, leaving about 9 m of free space from the nozzle, to allow 138 ballooning fibers to float horizontally without any adhesion to other objects. The wind speed and temperature were 139 measured with a PL-135 HAN hot-wire anemometer. To enable sampling of the ballooning fibers, the wind speed 140 was adjusted at 0.9 m s -1 , because spiders drifted downstream if the wind speed was above 1 m s -1 . The room 141 temperature was 22°-25°C. The ballooning behavior was stimulated with a 1000 watts hair dryer (low wind speed 142 mode) that produces warm air (28°-33°C) and the fluctuation of wind. The hair dryer was positioned beneath the 143 nozzle of the wind tunnel upward to avoid direct exposure to hot wind from the hair dryer (see S4). As soon as 144 spiders showed tiptoe behavior, the hair dryer was turned off. The turbulent intensity of the wind tunnel was 1.1% 145 (without the hair dryer) and 11.3% (when the hair dryer turned on). There was difference in temperature between 146 the laboratory and the field. The difference can be explained as follows: First, the ballooning behavior is coupled resources, etc [2,4,26,30]. If the biological pressure, for spiders to disperse, is high, spiders may try to disperse 149 even though it is low temperature. Second, the sudden increase of temperature acts on ballooning behavior as an 150 influential factor [31][32][33][34][35]. If there is the sudden increase of temperature, e.g. because of sunshine in the morning, 151 the ballooning behavior can be triggered even in low temperature condition. There are some reports that spiders 152 ballooned also at relatively low temperature, 10°-20°C [33,34; the author's field observation (13°-19°C)]. 153 Ballooning fibers were collected on a microscope slide, on which two narrow strips of a double-sided bonding 154 tape were attached. The ballooning fibers were sampled near the spinnerets (see Fig. 1B). A total of 11 samples 155 were prepared from 28 spinning events of ballooning silks. (In the experiment, a total of four spiders responded to 156 show ballooning behavior. Two of them were very active.). Two samples were selected, because the other 9 samples 157 failed to capture all the ballooning fibers on a single microscope slide or the fibers on the slide were deranged 158 during the capturing process. Simultaneously, silk fibers were captured on a square wire frame and carefully wound 159 around it in order to measure the length of ballooning threads (see Fig. 1C). The length of the silks was calculated 160 by multiplying the total number of half revolutions by the width of the square wire frame (20 cm). The successfully 161 sampled ballooning fibers were later studied with a field emission scanning electron microscope. The sampled ballooning fibers were coated with gold using a sputter coater (SCD 030, Balzers Union) and 165 observed with a field emission scanning electron microscope (DSM 982 Gemini, ZEISS, with 5-10 kV 166 accelerating voltage). The number of ballooning fibers was carefully counted and the thickness of fibers was 167 measured. The spinnerets of a female Xysticus cristatus, were also observed with the FESEM. For sample 168 preparation, the female spider was fixed in 2.5% glutaraldehyde and dehydrated in ascending concentrations of 169 ethyl alcohol from 30% to l00% (10 min at each concentration). After dehydration, the sample was dried with a 170 critical point dryer (CPD 030, BAL-TEC). The prepared sample was coated with gold using a sputter coater (SCD 171 030, Balzers Union) and observed with the FESEM (SU8030, Hitachi, with 20 kV accelerating voltage). 172

Investigation of the aerodynamic environment 173
For the investigation of the turbulent atmospheric boundary layer, ultrasonic three-dimensional wind speed 174 measurement took place on a grass field (53° 11' 42" N, 12° 09' 40" E, see Fig. 2A, B). This place is also a habitat 175 of the Erigone and Xysticus genus which do also ballooning behavior. To avoid mechanically induced updrafts by 176 hills, trees and rocks, a flat grass field was selected. The three-dimensional wind speed data at different two mean 177 Each adult and subadult crab spider (Xysticus genus) was raised separately in a plastic box (length × width × height: 186 13 × 13 × 7 cm), which has ventilation holes. Once a week, the spiders were fed a mealworm, Tenebrio molitor, 187 and moisture was provided with a water spray. 188

Ethics 189
The species used in the experiments (Xysticus genus) are not endangered or protected species. No specific 190 permissions were required. All applicable international, national and institutional guidelines for the care and use 191 of animals were followed.  In the Thomisidae family, not only female but also male adult spiders showed ballooning behaviors (see S1). 197 During the observation days, the temperature was 16°-19°C and the mean wind speed was 6-7 m s -1 (gust 14-17 198 m s -1 ) as reported by the nearest weather station in Dahlem. The sensor was installed on 36 m position above the 199 ground. Therefore, the local wind speed at 1.2 m above the ground must be much lower than these values. Later, 200 we checked the wind speed on a similar day, on which the mean wind speed from the weather station showed 6-7 201 m s -1 . The mean wind speed for 10 min showed 2.11 m s -1 . 202 On the experiment day, the spiders mostly showed tiptoe behavior. At the first stage of tiptoe behavior, the crab 203 spider evaluated the wind condition, not just passively through the sensory hairs on its legs, but rather actively, by 204 raising one of its front legs (leg I) or sometimes both, and waited in this position for 5-8 sec (see Fig. 3A, B, C). 205 This sensing behavior was often repeated a few times before the tiptoe pose. After each sensing step, the crab 206 spider rotated its body in the direction of the wind (see Fig. 3C, D). 207 If the spider decided that the wind was adequate to balloon, it raised its abdomen (already known as a tiptoe 208 behavior, see Fig. 3D) and spun its ballooning silks without any help from its legs. Before spinning ballooning 209 silks, there was a motion of a rear leg (leg IV) (see S5), with which the spider holds its safety line that connected 210 its spinnerets to the substrate, and then put it on the substrate (see S2A, B). 211 The crab spider first spun a single or a few number of fibers, and then many fibers (see wind condition was not appropriate, the spider cut the silk fibers and spun them again. If the ballooning silks 216 generated enough drag, the spider released the substrate and became airborne (see Fig. 4H). From careful video 217 investigation, it was observed that spiders stretched their all legs outwards, as soon as the spiders achieved takeoff. 218 Many ballooned crab spiders soared diagonally upwards along the wind flows. This paths had 5-20 degrees 219 inclination above the horizon. Some spiders traveled quasi-horizontally. Some spiders soared along a steep path 220 (about 45 degrees). During this steep takeoff, the spiders took off with relatively slow speed. The anchored drag 221 line (safety line) between the platform and the spider's spinneret could be seen. This anchored line endured without 222 breaking, until it became 3-5 meters long. After a while, it was broken mechanically. From the wind tunnel 223 experiment, it was found that the anchored line consists of two fibers. 224 Three new facts about ballooning were uncovered. First, the crab spider does not evaluate the wind condition 225 passively, but actively, by raising one of its front legs (leg I). Second, this adult ballooner anchors its drag lines on 226 the platform not only during its rafting takeoff, but also during tiptoe takeoff. Third, the crab spider postures all its 227 legs outwards and stretched, when airborne, not only at the takeoff moment, but also during the gliding phase (see 228 S6A-D). 229 Rafting pre-ballooning behavior was also observed. The local weather condition was a little bit colder and windier 231 than that of the previous observation day of tiptoe takeoff. Crab spiders were not active on that day. As soon as 232 they were set on the platform, they showed one of two behaviors. Either they hid on the opposite side of the 233 platform to avoid the wind, or they quickly retreated downwards about 0.4 to 1.1 m relying on their drag lines (see 234 S3A, B), and spun their ballooning fibers downstream of the wind. At this time, spiders spun a single or a few 235 number of fibers first, and then many fibers, as they showed during the tiptoe takeoff (see S3C, D). During this 236 process, the spiders also postured all their front legs and second legs outwards and backwards, so that they hung 237 The initial behaviors were mostly started with sensing motion. The frequent transitions between different behaviors 247 were occurred between sensing motion and tiptoe motion (see Fig. 5A). Spiders flew from only either the tiptoe 248 pose or the dropping and hanging pose (see Fig. 5B). The probability of the ballooning takeoff from tiptoe behavior 249 was 9.5%. The probability of the ballooning takeoff from the rafting pose was 37.5%. 250

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The duration of each tiptoe behavior was measured and their frequencies were analyzed. Short period tiptoe poses, 252 which lasted for less than 5 sec, were the most frequent. The longest tiptoe event lasted 65 sec. Successful 253 ballooning takeoffs were not biased in relation to tiptoe duration (see Fig. 6). 254

Gliding 256
A total of 32 floating threads were observed at the Teltow canal. Most of them were horizontally transported along 257 the channel at about 1-8 m above the water surface. They drifted passively due to light wind, but rarely fell down. 258 Some of their silks were inclined downstream. The others were inclined upstream. Two of 32 floating threads were 259 just threads alone without a spider. The number of observed threads was one to five. However, as not all threads 260 were visible with the naked eye, some may have been the multiple threads, which stuck together, although they 261 seemed to be a single thread. Some of them may not have been seen, because of their inappropriate angles and 262 positions in relation to the sun. Most of the spiders were positioned at the lower end of their threads. Although the 263 threads showed different numbers and shapes, they were usually laid diagonally (see Fig. 7). 264 265

Identification of ballooning fibers 266
The separation of glands for a drag line and ballooning lines was observed in ballooning behaviors in the laboratory. 267  The successfully collected ballooning fibers of both Xysticus cristatus and Xysticus species were observed with 274 the FESEM. Ballooning fibers consisted of two thick nanoscale fibers that were attached together (see Fig. 9B, C) 275 and many thin nanoscale fibers (see Fig. 9A-D). The two adult spiders, Xysticus species, spun, 48 to 58 thin nano-276 fibers and 2 thick nano-fibers (see Table 1

Aerodynamic environment on the short grass field 286
Two data sets at the different mean wind speeds (1.99 m s -1 and 3.07 m s -1 ) for 5 min were collected. Usable 287 updrafts were investigated in the turbulent atmospheric boundary layer. Each of the cases showed the vertical 288 deviation of 0.225 m s -1 and 0.267 m s -1 , respectively, which ranged from -0.5 to 0.5 m s -1 and from -0.6 to 0.7 289 m s -1 (see Fig. 10A, B). The turbulent intensities were 21 % and 23.7 % in the mean wind speeds of 1.99 m s -1 and 290  drag line. If there is a breeze, the drag line near its point of attachment to the platform would be sheared and drift 355 through the air [28,50]. From this context, many previous studies regarded that spiders use their drag line for 356 ballooning dispersal [16,17,22,23,38]. Some experiments substituted a drag line for a ballooning line [18,19,21]. From our wind tunnel test, we found that the Xysticus genus uses tens of nano fibers (diameters of 121 to 323nm) 388 for their aerial dispersal (see Fig. 9A-D). The number of ballooning fibers and their lengths were identified. Based 389 on these measured values, the required updraft speed for the ballooning takeoff was calculated using modified  reduced. Therefore, the Reynolds number of spiders' silks during their flight is much smaller than 0.04, and the 428 spider's flight is dominated by low Reynolds number fluid-dynamics [58]. However, a large spider's body shows 429 much larger scales than those of a spider silk, not only in size, but also in weight. In a free-fall case of a spider 430 body without any silks, the Reynolds number is about 2300 ( / , 2 ⁄ ; a spider's body 431 is assumed as a 5 mm diameter sphere with 25 mg weight, the projected area of a sphere: 19. 6 , the coefficient 432  Fig. 10). Therefore, the phenomenon that spiders usually balloon in the low wind 460 speed regime (lower than 3 m s -1 ) could be explained with this organized structure in the atmospheric turbulent 461 flows above the ground. However, the frequency and duration of these updrafts are not well known.  diagonally stretched shape of ballooning fibers may be caused by the shear wind in the atmospheric boundary layer. 471 We think that this may be helpful for long-endurance ballooning flight, because horizontally stretched silks produce 472 more drag, up to a factor of 2, than vertically distributed shapes of silk, because of an anisotropic drag of silks in 473 a low Reynolds number flow [70] (see S9). 474 The observed facts, that spiders outstretch all legs outwards during their flight, is puzzling because of its small 475 drag ratio compared to the drag of a spider's ballooning silks. Suter concluded that when a spider uses a relatively 476 short length of silk, the influence of posture on its terminal speed is greater than when the silk is very long [19]. dimensions of fibers, we concluded that these ballooning silks are two of minor ampullate silks and multiple 493 aciniform silks, which are usually used in other species as wrapping silks. Xysticus species, however, used these 494 silks for their aerial dispersal. Spiders showed also interesting behaviors, active sensing motion, such as evaluating 495 the wind conditions before their ballooning behavior. This behavior may save spider's silk dopes, which can be 496 consumed during their takeoff trials. 497 Two major features in the physics of ballooning are suggested from the study. First, atmospheric shear flow may 498 be helpful for the high buoyant capability of a ballooning structure, because horizontally/diagonally stretched silks 499 produce more drag than vertically distributed shapes of silks. Second, spiders may use the updrafts that are induced that these updrafts are correlated with lower wind speeds. Therefore, this hypothesis is expected that can explain 502 the fact why spiders usually balloon when the wind speed is lower than 3 m s -1 . However, these suggestions should 503 be studied further for theoretical firmness. 504 Whether or not vertical wind speed and fluctuation of wind influence on spiders' evaluation processes for 505 ballooning, or why spiders stretch their legs outwards during their flight, are questions that still remain. These 506 could be interesting topics for future research.