Brian G. Gall1, Kari L. Spivey2, Trevor L. Chapman3, Robert J. Delph4, Edmund D. Brodie

1 The Indestructible Insect: Velvet Ants from Across the United States Avoid Predation by 2 Representatives from all Major Tetrapod Clades.4 Brian G. Gall1, Kari L. Spivey2, Trevor L. Chapman3, Robert J. Delph4, Edmund D. Brodie,5 Jr.5, Joseph S. Wilson667 1Department of Biology, Hanover College, 517 Ball Drive, Hanover, IN 47243, 8 [email protected]9 2Department of Biology, Missouri State University, 901 S. National Ave., Springfield, MO 10 6589711 3Department of Biology, East Tennessee State University, 325 Treasure Lane, Johnson City, TN 12 3760413 4Department of Natural Resources, U.S. Army Dugway Proving Ground, 5330 Valdez Circle 14 Dr., Dugway, UT 8402215 5Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322-5305 1617 6Department of Biology, Utah State University-Tooele, 1021 West Vine Street, Tooele, UT 18 840741920212223241 2526 Abstract27 To determine whether birds might prey on velvet ants native to the eastern United States 28 (Dasymutilla vesta & Dasymutilla occidentalis), we trained birds to eat wax-worms from 29 feeders. We then exposed these birds to mealworms painted to resemble velvet ants, clay models,30 or live velvet ants, and recorded their behavior. Eastern bluebirds and mockingbirds readily 31 learned to eat from the feeders and both species consumed mealworms painted tan but avoided 32 aposematically painted mealworms. Mockingbirds also attacked clay models painted black but 33 would not strike at models painted to resemble a velvet ant. Trials with live velvet ants elicited 34 strong avoidance behavior by mockingbirds and no individuals would approach the dish. 35 Bluebirds that were presumably naïve did strike at live velvet ants, however none were 36 consumed and all velvet ants were unharmed. The results of these experiments suggest that birds 37 may be a limited threat to these extremely well-defended organisms.383940 Introduction41 Predation is an extremely powerful selective force driving the evolution of morphology, 42 physiology, and behavior among animals (Brodie et al., 1991; Endler, 1986; Lima and Dill, 43 1990). Because of the intense nature of the interaction (prey either escape to live another day or 44 die), it has resulted in a bewildering array of defensive structures and strategies to mitigate this 45 risk. Extreme examples include venomous frogs (Jared et al., 2015), salamanders with ribs that 46 pierce their skin (Brodie et al., 1984; Nowak and Brodie, 1978), beetles with rear rotary turrets 47 ejecting toxins at 100°C (Aneshansley et al., 1969; Arndt et al., 2015), and ouabain resistant 2 48 rodents with skeletons designed to take a punch (Kingdon et al., 2011). Regardless of the 49 defensive strategies utilized by prey, each is used during one of two distinct stages along the 50 chain of a predatory interaction (Endler, 1986; Hopkins et al., 2011); either before a predation 51 event has been initiated (predator-avoidance behavior) or after a predator has detected the 52 presence of its prey (antipredator mechanisms) (Brodie et al., 1991).53 Despite prey being well-defended, predators must eat, and a similar diversity of 54 mechanisms have evolved to help predators acquire their prey. For example, the terminal scales 55 on the spider-tailed viper (Pseudocerastes urarachnoides) have evolved to be flexible and is uses56 caudal luring to attract insectivorous birds which it envenomates and eats (Fathinia et al., 2015). 57 The lower jaw of dragonfly naiads has evolved into a protrudable grasping mouthpart allowing 58 the sit-and-wait predators to strike at prey with amazing speed (Needham and Westfall, 1954). 59 Apart from some apex predators, few organisms are likely to completely escape predation, and 60 even those prey which are so well-defended that it seems nothing can eat them have been found 61 to have a specialized predator (e.g. Brodie, 1968). One organism that possesses a myriad of 62 defensive structures and behaviors and for which its risk to potential predators is largely 63 unknown are velvet ants (order: Hymenoptera; family Mutillidae). Velvet ants are members of a 64 unique wasp family, Mutillidae. Their common name stems from the extensive setae that can 65 cover their entire body (fig 1) and the fact that the females are wingless, making them look like 66 ants. Although the taxonomic relationships within this group are beginning to be unraveled 67 (Williams, 2012), very little is known about their ecology (cite). Velvet ant females spend much 68 of their time actively searching for the nests of ground-nesting bees and wasps (cite). After 69 finding a host’s nest, the female velvet ant deposits an egg on or near the host pupae, which the 70 larvae consume after hatching (cite). 3 71 Given their flightlessness, one would expect diurnal females of this group to be highly 72 susceptible to predation. Yet, velvet ants have a number of defenses at their disposal to thwart 73 potential predators. Like other Aculate wasps, females are armed with a venomous sting, though 74 unlike most other wasps, velvet ants sting can be nearly half the length of their body. Although 75 the composition of the venom is unknown, it can be extremely painful (Schmidt, 1990; Schmidt 76 et al., 1984; Starr, 1985), which is often evident in their common names (e.g. cow killer). On a 77 human pain index, at least one velvet ant (Dasymutilla klugii) outscored 58 species of wasps and 78 bees in the painfulness of its sting, falling short of only the bullet ant, warrior wasp, and tarantula79 hawk in the amount of pain induced (Starr, 1985). The venomous nature of the females is 80 complemented by the striking aposematic coloration present of almost all diurnal species (Fig. 81 1). This coloration comes in various shades of white, orange, yellow, or red. Different 82 colors/patterns corresponds to a specific Müllerian mimicry ring consisting of dozens of species 83 (Wilson et al., 2012). These rings are extensive, with eight distinct rings making up one of the 84 largest Müllerian mimicry complexes on earth (Wilson et al., 2015)85 Although being dangerous and advertising this danger with bright colors is an obvious 86 antipredator mechanism, velvet ants possess several other defensive structures and behaviors. 87 When distressed, a stridulatory organ on their abdomen is contracted which produces audible 88 squeaking (Schmidt and Blum, 1977; Tschuch, 1993), and an alarm secretion may be released 89 from the mandibular gland (Fales et al., 1980; Schmidt and Blum, 1977). These function as 90 auditory and chemosensory aposematism, warning potential predators that if they continue an 91 attack, a sting is imminent. The exoskeleton of velvet ants possesses two properties that make it 92 an effective defense against predators. First, the exoskeleton is remarkably strong. Using a force 93 transducer, Schmidt and Blum (1977) calculated 11 times more force was needed to crush the 4 94 exoskeleton of a velvet ant as opposed to a honeybee (Apis). The rounded shape of the 95 exoskeleton also renders attacks more difficult as attempted stings or bites glance off the 96 abdomen instead of piercing it (Schmidt and Blum, 1977). 97 Despite the suite of defenses possessed by velvet ants (primarily females), relatively little98 is known about their relationships with potential predators or the pressures that may have driven 99 the evolution of these defenses. Schmidt and Blum (1977) conducted a series of studies with 100 Dasymutilla occidentalis and various potential predators. In this seminal work, ants, spiders, 101 lizards, and gerbils were presented velvet ants. Yet, only 2 out of 59 presentations resulted in the 102 consumption of a velvet ant by any predator; once by a tarantula and another by a gerbil. In most 103 cases, the velvet ants were either ignored from the start, or, were attacked, released, and 104 eventually left unscathed.105 Given the limited information on potential predators of velvet ants, we conducted a series106 of observational and experimental studies with a host of potential invertebrate and vertebrate 107 predators. The predators were selected based on the potential for natural interactions, the high 108 probability of contact with diurnal velvet ants, and dietary overlap (i.e. insectivorous). The 109 predators include: a praying mantis, toads, lizards, birds, shrews, and a mole. Experiments were 110 conducted with velvet ants from both the eastern and western United States (i.e. multiple 111 mimicry rings), with predators selected that are representative of the appropriate region. Because112 the predators and velvet ants used in all experiments were wild-caught, the experience of each is 113 generally unknown. 114 115 Methods116 Birds5 117 General Description118 All experiments took place in a manicured yard (0.4 ha) located in a rural setting near 119 Hanover, Indiana. Two feeding stations were attached to previously established bluebird nesting 120 boxes. Each feeding station consisted of a 15 cm diameter petri dish glued to the box such that 121 birds naturally perching on top of the box would see the dish and inspect the contents. Two 2 MP122 digital trail cameras (Wildgame Innovations) were either affixed to a post approximately 1 m 123 from the feeding station or were mounted directly to the box.124 Birds were initially trained to forage at the feeders by placing four wax moth larvae 125 (Galleria mellonella) in each petri dish at 0700 hrs each day for one week until testing began. 126 Days in which the birds were fed wax moth larvae will henceforth be called “training” days. The 127 photos from the trail cameras were obtained on the third day of training and reviewed to ensure 128 that the birds were feeding from the dishes.129 On “test” days, behavioral observations were conducted from a deck located 10 m and 20130 m from each of the respective feeding stations. A Nikon spotting scope (15-45x) and Bushnell 131 7x50 handheld binoculars were used to observe the feeding stations. The general procedure on 132 test days consisted of placing the appropriate experimental subject (see below) in the petri dishes133 at 0700 hrs and recording observations for 40 minutes. We recorded the species of bird visiting 134 the feeder, the general behavior of the bird toward the subjects in the dish, the type (control or 135 experimental animal) and number of experimental subjects struck at, and the type and number of 136 experimental subjects consumed. A minimum of two training days followed each test day. We 137 exposed wild-birds to the following treatments to determine if birds are potential predators of 138 velvet ants (experiments were conducted in the order presented): (1) mealworms (Tenebrio 139 molitor) or preserved female velvet ants (Dasymutilla vesta), (2) mealworms painted tan or with 6 140 the aposematic coloration of Dasymutilla occidentalis, (3) clay models painted black or with the 141 aposematic coloration of Dasymutilla occidentalis, or (4) live velvet ants (Dasymutilla 142 occidentalis). Dasymutilla occidentalis and Dasymutilla vesta are both members of the Eastern 143 mimicry ring, and therefore, have very similar coloration patterns.144145 Preserved Velvet Ants146 To determine if birds are potential predators of velvet ants, we exposed wild-birds to 147 either mealworms (Tenebrio molitor) or preserved female velvet ants (Dasymutilla vesta). 148 Pinned velvet ants were collected between 1951-1970 and were provided by the Utah State 149 University Insect Collection. The velvet ants were rehydrated by placing them in a sealed plastic 150 container on paper towels moistened with tap water. After 48 hrs, the velvet ants were removed 151 from the containers and the limbs, head, and antennae were repositioned so that the velvet ant 152 appeared in a normal crawling posture. After re-positioning the velvet ants, they were re-pinned 153 to polystyrene foam and left to dry for several days.154 On test days, a feeding station was randomly assigned to receive either four velvet ants or155 four mealworms. Pins were removed from the velvet ants before placing them in the feeder. Only156 complete specimens (i.e. not missing appendages or antennae) were used during the experiment. 157 A total of four replicates were conducted on separate test days.158159 Painted Mealworms160 Four mealworms were painted tan and four mealworms were painted red and black to 161 simulate the aposematic coloration pattern of the velvet ant, Dasymutilla occidentalis (figure 2). 162 We used a non-toxic and water-soluble acrylic paint that did not prevent the mealworms from 7 163 moving normally. Two mealworms of each color pattern were added to each of the feeding 164 stations and observations recorded for 40 minutes. A total of two replicates on separate test days 165 were conducted.166167 Live Velvet Ants168 The final experiment involved testing the responses of birds to live velvet ants. Two 169 female velvet ants (D. occidentalis) were collected near Hanover, IN and were housed in a 7.5 L 170 plastic tank with a screen lid. A 2 cm layer of sand was placed in the bottom of the tank. A dried 171 leaf (Magnolia grandifloria) was placed in the tank to provide cover. Fresh grapes were cut in 172 half and placed in the tank to provide a source of water and sugar until testing (1 week). To 173 ensure the velvet ants did not escape from the feeding dishes, we attached glass preparation 174 dishes (11 cm diameter x 4 cm deep) to the feeding stations. The velvet ants were removed from 175 the holding container by pushing them into a 25ml centrifuge tube and dumping them directly 176 into the glass dish; this procedure was used to ensure the velvet ants were not exposed to a 177 simulated predation event (i.e. grasping with forceps). Each feeding station had one live velvet 178 ant. At the completion of testing, mealworms were placed in the glass dishes to ensure the birds 179 were hungry. All mealworms were consumed shortly after being presented. One replicate was 180 conducted.181182 Mole183 A mole (Scalopus aquaticus) was collected in 2014 in a field on the Hanover College 184 campus. Fresh burrows were monitored during the morning and evening, and a dog (Canis lupus;185 terrier) was used to initially locate moles in their burrows. Upon detecting a mole, a researcher 8 186 removed the mole with a shovel and transported it to the lab in a 19-l container. Housing 187 chambers were designed so that there was a designated feeding area adjacent to a burrowing 188 chamber. The large section of the housing unit consisted of a container (55 × 35 × 30 cm) filled 189 with 20 cm of dry soil for burrowing. A feeding chamber (18 × 10 × 10 cm) was attached to the 190 burrowing chamber with a PVC tunnel (20 cm long, 5 cm diameter). The feeding chamber did 191 not contain soil, and any soil displaced into it by the mole was removed and placed into the 192 burrowing section. Moles were fed moist cat food every 24 hours.193 For testing, the mole was transferred to a test arena consisting of two chambers (11 × 11 194 × 16 cm) connected by a clear tube (30 cm long, 6 cm diameter). The arena was left empty. After195 transferring the mole to the test arena, a 5-minute acclimation period was initiated. Following the196 acclimation period, a velvet ant was introduced into the arena and observations were recorded for197 25 minutes. At the conclusion of the trial, a control cricket was introduced and immediately 198 consumed.199200 Shrews201 Shrews (Blarina brevicauda, n = 4) were collected on 17 November 2014 in a wooded 202 area on the Hanover College campus. Sherman live traps (HB Sherman Traps, Inc.) were baited 203 with wet cat food and were placed in close proximity to burrows and logs throughout the forest. 204 Traps were checked every three hours and captured shrews were placed in a 19-l container and 205 transported to the lab. Individuals were housed in 38 L chambers with a 2-inch layer of dry soil 206 (collected from campus), strips of cotton cloth, and a water dish. Shrews were maintained on a 207 diet of moist cat food and fed every 24 hours. A single shrew (Notiosorex crawfordi; henceforth 9 208 “UT shrew”) was collected from Dugway Proving Ground, Utah, and housed under similar 209 conditions.210 For experimental trials, the shrews were placed in 38 L aquaria that were completely 211 empty. The shrews were allowed to acclimate for 5 minutes, after which a velvet ant was 212 introduced. Detailed observations were then recorded for approximately 20 minutes, after which 213 the velvet ant was removed and a control cricket was introduced into the chamber. For the 214 experimental trial with the UT shrew, the velvet ant’s stinger was removed with forceps. Shrews 215 were tested only once and were given a control cricket at the completion of the trials. All control 216 crickets were immediately consumed.217218 Toads219 A single American toad (Anaxyrus americanus) was collected on Hanover College’s 220 campus and housed in a 38 L aquaria with damp sphagnum moss. The toad was not fed until 221 testing (2 days). For testing, the American toad was transferred to an empty 38 L tank and 222 presented a velvet ant for 20 minutes. At the conclusion of testing, the toad was presented, and 223 immediately consumed a cricket.224 Two Great Basin spadefoot toads (Spea intermontana) were collected from Dugway 225 Proving Ground and housed individually in 150 L tanks. Each toad was presented (in its home 226 tank) a velvet ant (either Sphaeropthalma mendica or Dasymutilla scitula) on two separate 227 occasions. The testing days were separated by at least 3 days. After each trial the toads each 228 consumed a cricket.229230 Lizards10 231 Between 2013 and 2015, we collected lizards [Aspidoscelis tigris (n = 6), Uta 232 stansburiana (n = 3), Gambelia wilzenii (n = 2) from Dugway Proving Grounds, UT, to test the 233 antipredator defenses of various species of velvet ant. Lizards were collected with pitfall traps 234 and housed in 227 L tanks with sand substrate, a water dish, and various natural elements (sticks,235 rocks, etc.). Each tank had a UVB daytime heat lamp (Exo Terra) and a heat rock (24 hrs). 236 Lizards were fed commercially available crickets (Acheta domestica) and mealworms (Tenebrio 237 molitor) ad libitum. Prior to testing, lizards were in captivity between 2 weeks to 2 years, with 238 most between 4-12 months. Food was withheld from each lizard for 3 days prior to testing. The 239 responses of each lizard to velvet ants were conducted in the lizard’s home tank to reduce 240 handling effects. On test days, trials were conducted at 0800 hrs and consisted of a single velvet 241 ant (various species, Table 2) being dropped into the tank. Observations were recorded for 5 min,242 after which the velvet ant was removed and a control cricket was introduced. Each lizard quickly243 consumed a control cricket at the completion of the trial. In addition to each initial trial with a 244 lizard, a series of “secondary” trials were also conducted with various species of velvet ants. 245 These trials were conducted at least one day following each primary trial. Results of the 246 secondary trials are discussed separately from the initial trials (see below).247 In addition to the predation trials conducted in captivity, two semi-natural trials were 248 conducted. In the first, a velvet ant (Dasymutilla scitula) was placed in a glass dish (with lid) and249 set in the open in a sandy area LOCATION. In a second trial, a velvet ant (Dasymutilla foxi) was250 tied to a small thread and staked in the ground in an open area LOCATION. Observations were 251 recorded for 1.5 hrs from approximately 10 m away.252253 Praying Mantis11 254 A European mantis (Mantis religiosa) was collected on 30 September 2013 on the Utah 255 State University – Tooele campus and housed in a 3.8L glass aquarium with a screen lid. A 2 cm 256 layer of sand was placed in the bottom of the tank and small rock and some twigs were added to 257 provide a place for the mantis to perch. A single velvet ant (Dasymutilla vestita) was placed into 258 the tank and no other food source was provided. The tank was sprayed with water every day to 259 provide water and the enclosure was placed near an east facing window so the insects would 260 receive morning sun. Observations were made daily to determine if the velvet ant had been 261 consumed. 262263 Results264 Birds265 Preserved Velvet Ants266 Mockingbirds were the only species to visit the feeding station during observations. At 267 least 4, and likely 5, separate mockingbirds were seen foraging at the stations throughout the 268 experiment (i.e. multiple birds were visible in the same field of view). Mockingbirds exhibited 269 significantly more strikes at mealworms (n = 16) than preserved velvet ants (n = 1; χ2 = 13.2, P <270 0.001). A single mockingbird did exhibit one strike at a preserved velvet ant, however it was 271 immediately dropped and not consumed. All strikes on the mealworms were immediately 272 followed by consumption (n = 6), or the mealworm was held in the beak and carried away from 273 the feeder (n = 10); in these cases the birds flew out of view and, although they were likely 274 consumed, their fate is unknown. If these mealworms are categorized as consumed, the 275 mockingbirds consumed significantly more mealworms (n = 16) than preserved velvet ants (n = 276 0; χ 2 = 16.0, P < 0.001).12 277278 Painted mealworms279 The mockingbirds consumed more tan painted mealworms (n = 4) than mealworms 280 painted with the Dasymutilla aposematic color pattern (n = 0; χ 2 = 4.0; P = 0.045). However, the 281 mockingbirds exhibited more strikes at aposematically painted mealworms (n = 13) than tan 282 painted mealworms (n = 5; χ 2 = 3.55; P = 0.06). Three of the four tan-colored mealworms were 283 consumed immediately by the mockingbirds. One mealworm was struck and dropped before 284 being picked up and consumed. Despite receiving more strikes than neutrally colored 285 mealworms, the mockingbirds appeared hesitant to feed on the aposematically painted 286 mealworms and none were consumed over the course of the trials. One bird tilted its head so as 287 to visually inspect the dish, got approximately 15 cm from the mealworm, and retained this 288 posture for 30 seconds. The bird then struck at an aposematic mealworm and carried it to the 289 ground 6 m from the feeding station. Later inspection found a damaged, but uneaten, aposematic 290 mealworm at this location. The mealworm had an “open wound” on the dorsal side of where the 291 head would normally be on a live velvet ant/mealworm. Another aposematic mealworm was 292 inspected, struck, and dropped a total of six times before being carried to the ground 293 approximately 20 m from the feeding station. The bird then appeared to peck vigorously at the 294 worm for several seconds before flying away. Later inspection of the site discovered a 295 mealworm that had been “decapitated” but which was otherwise unharmed and uneaten (Fig X). 296 No aposematically colored mealworms were consumed during any trial.297298 Live Velvet Ants299 Birds13 300 During trials with live velvet ants, mockingbirds appeared hesitant to visit the feeders. 301 The birds landed on top of the feeding station, glanced at the dish, and flew away. This behavior 302 had not been observed with any other trials; mockingbirds typically landed next to the dish and 303 inspected the contents before ignoring or striking the available prey. No strikes were exhibited 304 toward the live velvet ants by mockingbirds, however, control mealworms were immediately 305 consumed at the conclusion of the trial.306 In addition to mockingbirds, at least 5 separate juvenile bluebirds also visited one of the 307 feeding stations during the trial. On four occasions the birds landed on top of the station, 308 inspected the dish, but flew away without approaching. On one occasion, a bird landed next to 309 the dish, inspected the velvet ant, and flew away. A fifth bluebird landed on the edge of the dish 310 and struck a live velvet ant twice on the thorax. The velvet ant was visibly struck because it 311 became flattened against the bottom of the glass dish. However, the bird did not grasp the velvet 312 ant in its beak and, given the lack of visual distress, it is doubtful whether the bird was stung 313 during the interaction; it is unknown whether the velvet ant stridulated during the interaction.314315 Mole316 The mole attacked the velvet ant once during the interaction. After the initial attack, the 317 velvet ant appeared to escape unharmed and the mole did not appear to be stung by the velvet 318 ant. Shortly after, the velvet ant and mole passed through the central tube simultaneously and got319 “wedged” together inside the tube. After a few seconds, the mole began thrashing wildly and 320 appeared to be stung by the velvet ant. After retreating to opposite chambers, the mole began 321 rubbing the area where the velvet ant had previously been wedged and where the mole had 14 322 presumably been stung. After these initial interactions, the mole and velvet ant came in contact 4 323 separate times. Each time, the mole recoiled and rapidly retreated from the velvet ant.324325 Shrew326 After introducing a Dasymutilla vesta to a short-tailed shrew, the shrew vigorously 327 sniffed the velvet ant and struck it. However, the velvet ant was rejected. It is unknown if the 328 shrew was stung. The shrew rapidly moved about the chamber exhibiting escape behavior until 329 the end of the trial. During an interaction between a short-tailed shrew and a Dasymutilla 330 occidentalis, the velvet ant stridulated upon contact with the shrew 5 separate times. The velvet 331 ant was never attacked. In another trial with a short-tailed shrew, the velvet ant was attacked 7 332 separate times in the first five minutes of the trial. Each time the velvet ant stridulated and was 333 released. On the eighth attack, an audible crack was heard after which the velvet ant was flung 334 across the chamber and repeatedly attacked. After a series of attacks, the shrew paused and 335 appeared irritated. The right front paw was enlarged and the shrew continually licked and 336 chewed at this paw (presumably stung). At the completion of the trial the velvet ant was still 337 alive and was inspected for injuries. A small patch of setae was discolored on the abdomen, but 338 no puncture in the exoskeleton was visible. During the final shrew-velvet ant trial, the velvet ant 339 was bitten on the posterior portion of the thorax. An audible crack was heard during this strike. 340 The velvet ant immediately stridulated and the velvet ant was dropped; the shrew did not appear 341 to be stung. Shortly after, the velvet ant was struck again, during which the shrew was stung in 342 the mouth and the velvet ant was dropped. The velvet ant was attacked 6 separate times after this343 event. After these attacks, the velvet ant’s stridulations became inconsistent and could not walk. 344 The shrew began itching its head and side of the neck vigorously, as well as biting its right-front 15 345 paw. Any further contact between the velvet ant and shrew resulted in avoidance. All velvet ants 346 survived the interactions with shrews.347 When a velvet ant (Dasymutilla bioculata – sting removed) was introduced to the UT 348 shrew, it immediately attacked the velvet ant, dropped it after the velvet ant stridulated, and ran 349 to the opposite side of the test chamber. The velvet ant’s exoskeleton was slightly cracked, but 350 the velvet ant survived the interaction.351352 Toad353 When presented with a velvet ant (Dasymutilla occidentalis), the American toad hoped 354 toward the velvet ant and upon contact inflated its lungs, dropped a shoulder, and closed the eye 355 closest to the velvet ant. The toad remained in this position until the velvet ant was removed (~20356 min).357 Upon the initial interactions with a velvet ant, each Spadefoot toad attacked and 358 swallowed a velvet ant. However, in each case the velvet ant was quickly regurgitated, which 359 was followed by the toad wiping its hands over its tongue multiple times. Both velvet ants were 360 unharmed. During the second set of interactions, both toads avoided the velvet ants completely.361362 Lizards363 Among the three species of lizards, and 12 independent trials, only two lizards (one 364 whiptail, one side-blotched lizard) attacked a velvet ant (Table 3). In each case the lizard was 365 stung in the face and quickly dropped the velvet ant, after which it avoided the velvet ant. The 366 velvet ants were unharmed in each case. Twenty-four hours following the initial trial with the 367 side-blotched lizard described above, the animal was found dead in its tank with a noticeable 16 368 discoloration on the head where it had been stung. The remaining lizards either ignored the 369 velvet ant completely (n = 4), or approached the velvet ant (n = 6). Approaching the velvet ant 370 was followed by avoidance (n = 2), tongue flicking (n = 1), or nudging the velvet ant with their 371 snout (n = 3). In 59 secondary trials with these same lizards, only 4 strikes were exhibited. In 372 each case, the lizard was one that had not previously struck a velvet ant (i.e. had not been stung). 373 One strike by a leopard lizard resulted in the lizard swallowing the velvet ant. However, the 374 lizard immediately regurgitated the velvet ant and exhibited avoidance; it is unknown if the 375 lizard was stung on the inside of the mouth. Across all 71 trials, no velvet ant was injured or 376 killed during an interaction with a lizard.377 While most secondary trials (where lizards that had previously been exposed to velvet 378 ants) took place within a week of the initial trial, the one whiptail lizard that attacked the velvet 379 ant and was stung in the face was re-exposed to a velvet ant 15 months later. This whiptail 380 closely watched the velvet ant, but did not attempt to attack it.381 In the semi-natural trials, a single lizard (Aspidoscelis tigris) approached the glass dish, 382 nudged the lid off the dish and grabbed the velvet ant. It then immediately ran under a nearby 383 bush, dropped the velvet ant, and ran away. The velvet ant was observed crawling into a burrow 384 under the bush and neither the velvet ant or lizard were recovered. In the second trial, a single 385 lizard approached the snared velvet ant, tongue flicked it several times, and then avoided the 386 velvet ant.387388 Praying Mantis389 No observed attacks on the velvet ant were made until October 10, 2013. At 390 approximately 1100 hrs the velvet ant was observed sitting still on a rock sunning itself when the17 391 mantis approached slowly and grabbed it. The mantis immediately attempted to bite the velvet 392 ant on the thorax but it looked like the mantis was unable to bite through the velvet ants cuticle 393 on the edge of its thorax (this was later confirmed by examining the velvet ant under a 394 microscope and there was no visible sign of damage of any kind). As soon as the mantis grabbed 395 the velvet ant, the velvet ant began attempting to sting the mantis but it looked like the sting 396 couldn’t easily penetrate the cuticle of the mantis' forelimbs. The velvet ant also began 397 stridulating and continued to do so even after it was released. The mantis held the velvet ant for 398 about 5 seconds then suddenly released it. It was unclear if velvet ant was able to sting the 399 mantis but following the release of the velvet ant the mantis began grooming itself for about 5 400 seconds, focusing primarily on the arm that was closest to the velvet ant’s sting. About 15 min 401 after the encounter with the velvet ant the mantis was offered a moth, which it immediately 402 consumed. Approximately 24 hours after the trial with the velvet ant, the mantis was offered a 403 yellow jacket (Vespula vulgaris), which it immediately consumed.404405 Discussion406 The results of this study indicate that velvet ants from both the Eastern and Western 407 United States possess a myriad of defenses that render them almost invulnerable to a suite of 408 potential predators including an insect, amphibians, reptiles, birds, and small mammals. The 409 predators selected were chosen based on a high probability of interaction and dietary overlap that410 would make interactions between these species highly likely in the wild. Nevertheless, out of 411 over 100 interactions between potential predators and various species of velvet ant, velvet ants 412 were struck at only 15 separate times and the three velvet ants that were eaten were immediately 413 regurgitated.18 414 The birds that visited our feeders during this study forage heavily on insects (Beal, 1915; 415 Cottam and Knappen, 1939), yet all birds appeared extremely wary around both live and dead 416 velvet ants, as well as models and mealworms painted to resemble velvet ants. These same birds 417 foraged immediately upon control mealworms. A similar avoidance response was observed by a 418 single starling (Sturnus vularis) in a trial by Schmidt and Blum (1977). While the experience of 419 our birds is unknown, work with the aposematic color patterns of snakes indicates that these 420 patterns (red/yellow/black) are avoided by avian predators (Brodie, 1993; Brodie and Janzen, 421 1995) and that this avoidance is innate in at least one species of neotropical bird (Smith, 1975). 422 Studies with invertebrate prey are more ambiguous and both innate and learned avoidance of 423 aposematic patterns has been observed (Coppinger, 1970; Exnerová et al., 2006; Svádová et al., 424 2009). The bluebirds visiting our feeders had recently fledged (juvenile plumage; likely the same425 birds that had recently fledged from the box making up the feeding station). Yet, with the 426 exception of one strike, even these young birds avoided the velvet ants. Interestingly, mealworms427 painted with aposematic coloration matching velvet ants did receive more strikes than plain 428 mealworms and two were decapitated but left uneaten. Partially eating or seizing and pecking at 429 newly discovered distasteful prey occurs in some birds (Wiklund and Järvi, 1982), and these 430 results suggest the mockingbirds were experienced with insect warning coloration but may not 431 have had prior experience with velvet ants.432 Similarly to birds, various species of lizards were wary around the velvet ants and no 433 velvet ant was injured or eaten by these lizards out of 71 total interactions. Even in field trials 434 with tethered velvet ants, none were consumed. These results were surprising given the diurnal 435 activity patterns, stout head and jaws, and insectivorous nature of the lizards tested. The natural 436 history of both predator and prey in this case likely brings both species into contact frequently, 19 437 yet lizards do not appear to be predators of velvet ants. Schmidt and Blum (1977) tested lizards 438 from Florida with local velvet ants and while some did attack, all velvet ants were released 439 unharmed. Similarly, two horned lizards (Phrynosoma cornutum), which regularly prey upon 440 unpalatable ants, consumed a cryptically colored Dasymutilla dilucida but ignored three species 441 of aposematic velvet ants (Manley and Sherbrooke, 2001). The broadhead skink (Plestiodon 442 laticeps) is the only lizard to have successfully consumed velvet ants during experimental trials. 443 These occurred after repeated failed attacks (up to 23), during which an interaction in the wild 444 would have likely resulted in the velvet ants successful escape (Vitt and Cooper, 1988).445 Predator avoidance and antipredator defenses are used at different points during 446 interactions between predators and prey. This sequence occurs from approach and identification 447 to the eventual subjugation and consumption of the prey (Endler, 1986; Hopkins et al., 2011). Of 448 the specific defenses present in velvet ants, each can function at different stages of the predator-449 prey interaction, thus maximizing the probability of surviving the interaction (as prey move 450 further along in the interaction the probability of survival decreases). In addition, the role of a 451 particular defense is also dependent on the particular predator type. For example, almost all the 452 birds and many of the lizards tested avoided the velvet ants immediately upon sight of the 453 warning coloration; birds and lizards are visually oriented predators (cite). While shrews are 454 well-known to be voracious predators (e.g. Brodie et al., 1979), they have poor vision (cite) and 455 all but one shrew attacked the velvet ants, many multiple times. In some of these cases, 456 stridulation (and possibly release of a chemical signal) was enough to cause the release of the 457 prey. However, in most cases the interaction escalated and envenomation was required to prevent458 predation; all shrews eventually exhibited avoidance.20 459 Velvet ants appear to possess an effective suite of defense mechanisms; a hard and 460 slippery exoskeleton, venom, warning chemicals and sounds, rapid escape behavior, and bright 461 coloration. While these are common defenses among animals (Endler, 1986), this combination 462 appears to make velvet ants almost immune to predation. The pressure to evolve this suite of 463 defenses was likely intense, and the diurnal and flightless nature of the females may have played 464 a role in this evolution. While the observations presented here provide strong evidence that these 465 adaptations function in defense, function is not always responsible for the form, and the 466 dangerous nature of their hosts must not be overlooked (Deyrup, 1988). Female velvet ants 467 parasitize ground dwelling bees and wasps (best citation?), and the size and strength of their 468 exoskeleton also prevents penetration by the biting and stinging insects they parasitize (Brothers,469 1972). Further, the relatively rare and scattered nature of the host nests requires females to spend 470 extensive time searching for hosts, leaving females vulnerable to predation throughout this 471 duration and possibly leading the evolution of some of these defenses (e.g. stridulations, venom) 472 (Deyrup, 1988).473 Schmidt and Blum (1977) suggest that velvet ants may have evolved different defenses in474 response to different predators. While that may be true, our observations indicate that it is the 475 combination of these defenses that enable velvet ants to be so successful. For example, we find 476 that when an inexperienced lizard first encounters a velvet ant and attacks it, the hard slippery 477 cuticle of the velvet ant stops the lizard from immediately crushing its intended prey. The lizard 478 then attempts to manipulate the velvet ant in its mouth, which gives the velvet ant time to sting 479 the lizard in the mouth. This painful sting causes the lizard to release the velvet ant, where it is 480 immediately exposed to both the aposematic colors and the striduations. The sting, accompanied 481 by the warning coloration and sounds appear to provide an effective deterrent to future predation 21 482 events, in our trials after one failed predation attempt a lizard still avoided the velvet ant even 483 after 15 months with no reinforcement of the signal.484 The extreme effectiveness of the velvet ant defensive suite has likely led to the evolution 485 of the large velvet ant mimicry complexes found throughout North America (cite). These 486 complexes include both harmless Batesian mimics as well as Müllerian mimics (cite). The 487 Batesian mimics include those of spiders (Edwards, 1984; Nentwig, 1985), antlion larvae (Brach,488 1978), and several beetles (Acorn, 1988; Mawdsley, 1994; Lanteri and Del Rio, 2005). Velvet 489 ants, along with some other wasps, form the largest known Müllerian mimicry complex 490 worldwide, with over 350 species from 25 genera and two families participating in eight distinct 491 mimicry rings (Wilson et al. 2012; 2015; Rodriguez et al. 2014).492493494 It has been suggested that because velvet ants are similar in appearance and behavior of ants, 495 predators that often eat ants could pose a threat to velvet ants (Pan et al. 2017). The evolution of 496 aposematism and long setae seen on many velvet ants, therefore, likely developed as a way to 497 differentiate themselves from true ants to reduce attacks from ant specialist predators. Many 498 diurnal velvet ants have bright contrasting colors and relatively long setae, which are not seen in 499 true ants. 500501502503 Summarize our results504 Discuss in relation to other studies on predators of velvet ants22 505 Suggest velvet ants are essentially invincible506 Evolution of these defenses507 combination of selection from predators (slow moving, flightless, diurnal)508 can’t rule out selection from host D – what paper was that – Michigan lake?509 any link to parasite host interactions?510 Likely why mimicry complexes so large and so many different ones exist511 Talk about joes other two papers?512 What limits populations – distribution and abundance of host513 Any other examples of this in nature?514 Surprising because studies show parasites are often responsible for regulating host515516 Acorn, J. H. Mimetic tiger beetles and the puzzle of cicindelid coloration (Coleoptera: 517 Cicindelidae). Coleopterists Bull. 42, 28-33 (1988).518 Mawdsley, J. R. Mimicry in Cleridae (Coleoptera). Coleopterists Bull. 48, 115-125 (1994).519 Lanteri, A. A. & Del Rio, M. G. Taxonomy of the monotypic genus Trichaptus Pascoe 520 (Coleoptera: Curculionidae: Entiminae), a potential weevil mimic of Mutillidae. 521 Coleopterists Bull. 59, 47-54 (2005).522 Edwards, G. Mimicry of velvet ants (Hymenoptera: Mutillidae) by jumping spiders 523 (Araneae: Salticidae). Peckhamia 2, 46-49 (1984).524 Nentwig, W. A mimicry complex between mutillid wasps (Hymenoptera: Mutillidae) and 525 spiders (Araneae). Stud. Neotrop. Fauna Envir. 20, 113-116 (1985).23 526 Brach, V. Brachynemurus nebulosus (Neuroptera: Myrmeleontidae): a possible Batesian 527 mimic of Florida mutillid wasps (Hymenoptera: Mutillidae). Entomol. News 89, 153-156 528 (1978).529 Rodriguez, J., J.P. Pitts, C.D. von Dohlen, and J.S. Wilson. 2014. Müllerian mimicry as a result 530 of codivergence between velvet ants and spider wasps. PLoS ONE. 9: e112942.531 Pan, A.D., K.A. Williams, and J.S. Wilson. 2016. Are diurnal Iguanian lizards the evolutionary 532 drivers of New World female velvet ant (Hymenoptera: Mutillidae) Müllerian mimicry rings? 533 Biological Journal of the Linnean Society. DOI: 10.1111/bij.12894534535536 Acknowledgements537 All animals from Utah were collected and housed under COR permit #4COLL8467. The 538 shrews and mole were collected under Indiana Scientific Purposes License #14-040.539540 Tables541 Table 1. Species of velvet ants tested with lizard predators, number of trials conducted for each 542 species, and the number of instances that species was attacked.# of # of Velvet ant species trials attacks Dasymutilla arenivaga 2 1 Dasymutilla bioculata 18 0 Dasymutilla foxi 8 1 Dasymutilla gloriosa 10 0 Dasymutilla gorgon 14 2 Dasymutilla scitula 9 1 Dasymutilla vestita 5 1 Sphaeropthalma mendica 4 1 54324 544 Table 2. Summary of the outcomes from initial and secondary trials with 4 species of lizards and 545 various velvet ants. Number in parentheses are ?.# of primary # of # of # of ants # of # of ants injured Lizard species trials investigations strikes consumed stings or killed Aspidoscelis tigris 6 5 (4) 2 (1) 0 2 (1) 0 Eublepharis macularius 1 0 0 0 0 0 Gambelia wislizenii 2 1 0 0 0 0 Uta stansburiana 3 3 1 0 1 0# of secondary # of # of # of ants # of # of ants injured Lizard species trials investigations strikes consumed stings or killed Aspidoscelis tigris 36 21 (19) 3 0 2 0 Eublepharis macularius 2 0 0 0 0 0 Gambelia wislizenii 15 4 1 1 0 0 Uta stansburiana 6 1 0 0 0 0 546547 Figures548 549 Figure 1. Painted clay models (left) and painted mealworms (right) used to test the role of 550 aposematic coloration found in Dasymutilla occidentalis during interactions with wild birds.55125 552553 Figure 2. Photograph of an aposematically painted mealworm that was struck at by a 554 mockingbird and “decapitated” but not consumed.55526 556557 Figure 3. 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Brian G. Gall1, Kari L. Spivey2, Trevor L. Chapman3, Robert J. Delph4, Edmund D. Brodie

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