A HUNTING STICKS TO THE WEBS OF ITS PREY

A Thesis

Presented to

The Graduate Faculty of the University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Master of Biology

Max Headlee

May 2019

i

A SPIDER STICKS TO THE WEBS OF ITS PREY

Max Headlee

Thesis

Approved: Accepted:

______Adviso r ______Todd Blackledge Department Chair Steve Weeks

______Committee Member Dean of College Randy Mitchell Linda Subich

______Dean of the Graduate School Committee Member Peter Niewiarowski Chand Midha

______Date

ii ABSTRACT

Chalybion californicum is a spider hunting wasp that utilizes an aggressive mimicry strategy in which it lands in the spider’s web, although its close relative and fellow spider hunter caementarium does not do this. This study tested three hypotheses to determine if C. californicum possesses unique anti-adhesion traits to allow it to hunt in its peculiar manner. First, I hypothesized that C. californicum has unique morphological traits that could reduce its adhesion to capture silk, but observations made with scanning electron microscopy revealed a lack of qualitative morphological differences between the tarsi of C. californicum and S. caementarium. Second, I hypothesized that C. californicum would adhere less strongly to capture silk than that do not actively land in spider webs. By measuring the work done to pull sticky silk off of wasp body parts, I found that C. californicum adhered to this silk with similar strength as several of these other wasp . Third, I hypothesized that C. californicum could escape entanglement in an orb web faster than wasps that did not specialize on web building , but after throwing these wasps into webs and measuring the length of time they took to free themselves, I found that C. californicum did not escape significantly more quickly than either of the other tested species. I conclude that C. californicum does not need unique physical anti-adhesion traits to make its web landing behavior a viable hunting strategy, and the differences in predatory behavior between it and its relative S. caementarium are more likely due to niche partitioning than physical differences between these species.

iii TABLE OF CONTENTS

Page

LIST OF TABLES ...... v

LIST OF FIGURES ...... vi

CHAPTER

I. INTRODUCTION ...... 1

II. METHODS ...... 5

Study Organisms ...... 5

Scanning Electron Microscopy ...... 7

Adhesion Trials ...... 8

Escape Trials ...... 11

Statistical Analysis ...... 13

III. RESULTS ...... 14

IV. DISCUSSION ...... 24

LITERATURE CITED ...... 30

APPENDIX ...... 35

iv LIST OF TABLES

Table Page

3.1 P-values of comparisons of abdomen and wing adhesion ...... 17

3.2 P-values of comparisons of mid femur and mid tarsus adhesion ...... 18

3.3 P-values of comparisons of hind femur and hind tarsus adhesion ...... 19

3.4 Paired comparisons of time to escape entanglement of wasps thrown into orb webs ...... 22

v LIST OF FIGURES Figure Page 3.1 Scanning electron microscope images of C. californicum tarsi (a) Fore tarsus (b) Mid tarsus ...... 16

3.2 Comparisons of adhesion of various body parts between species (a) Abdomens – Relative (b) Abdomens – Whole Part (c) Mid femurs – Relative (d) Mid femurs – Whole Part (e) Mid tarsi – Relative (f) Mid tarsi – Whole Part ...... 20 (g) Hind femurs – Relative (h) Hind femurs – Whole Part (i) Hind tarsi – Relative (j) Hind tarsi – Whole Part (k) Wings – Relative ...... 21

3.3 Comparisons of time to escape entanglement in an orb web between pairs of californicum (Cc) and (Sc) thrown into the web ...... 22

3.4 Comparisons of time to escape entanglement in an orb web between pairs of Chalybion californicum (Cc) and fuscatus (Pf) thrown into the web ...... 23

vi CHAPTER I

INTRODUCTION

An may possess a trait that incurs the evolution of further traits, such as a physical adaptation that facilitates the development of new behaviors (Tinbergen, 1963;

Ferry-Graham et al. 2002; York & Fernald, 2017). For example, different jaw morphologies in bower building cichlid fishes have influenced the evolution of different building styles in these species (York et al. 2015). Understanding why an animal has a particular behavioral trait may therefore require studying associated physical traits

(Tinbergen, 1963; Ferry-Graham et al. 2002).

Two species of wasps, Chalybion californicum and Sceliphron caementarium (), have a number of similarities and differences that make them useful organisms for studying the evolution of behavior. These species both provision their with paralyzed spiders to feed their young, and there is substantial overlap in their accepted prey ranges (Muma & Jeffers, 1945; Horner & Klein, 1979; Blackledge &

Pickett, 2000; Blackledge et al. 2003). This is particularly true of their araneid orb weaver prey, of which some species are hunted by both of the mud daubers (Peckham &

1 Peckham, 1898; Blackledge & Pickett, 2000). C. californicum does not construct its own nests, instead reusing ones made by S. caementarium, and consequently these species live in the same locations (Rau, 1928). In spite of their similarities, the wasps differ greatly in their hunting behaviors. S. caementarium flies at spiders and either attacks them directly or, sometimes in the case of web building species, induces them to flee the web onto the surrounding substrate where they may be more vulnerable to the wasp (Eberhard, 1970;

Blackledge & Pickett, 2000). C. californicum utilizes a strategy of aggressive mimicry: it lands in or near a spider web and plucks the threads to imitate the struggles of an ensnared , drawing the spider directly to it (Rau & Rau, 1918; Coville, 1976;

Blackledge & Pickett, 2000). While C. californicum’s hunting behavior may entail repeated close contact with sticky spider silk, this does not appear to typically hinder the wasp from getting out of the web (Coville, 1976). These wasps use their respective hunting strategies even when predating the same species of spider, raising the question of why these wasps evolved to behave so differently under similar circumstances

(Blackledge & Pickett, 2000).

Jeanne (1972) has suggested that wasps that exhibit the behavior of landing in spider webs utilize physical adaptations that prevent them from adhering to the gluey silk.

Anti-adhesion traits unique to C. californicum might help to explain why it actively enters webs while S. caementarium does not, but what physical traits could reduce C. californicum’s adhesion to viscid spider silk? Perhaps a useful reference for C. californicum would be to its spider prey, as they are also in frequent contact with viscid glue and manage to not adhere strongly. The setae of web building spiders have branches that snag on the threads of the web, preventing those threads from reaching the

2 underlying cuticle where the glue might be able to contact more of the body (Briceño &

Eberhard, 2012). Tarsal plantulae (extensions of the cuticle that aid in adhesion) are generally absent in the Chalybion , but present among Sceliphron species (Bohart &

Menke, 1972). In addition to these modified setae, a hydrophobic coating on the legs of spiders causes the glue to adhere less strongly in the places where it does manage to make contact (Briceño & Eberhard, 2012; Kropf et al. 2012). There is evidence that could similarly achieve reduced adhesion to capture silk via cuticular waxes and oils

(Nentwig, 1982; Betz & Kölsch, 2004 and references therein). Thus, there are indeed physical traits that could be used to weaken adhesion to sticky spider silk.

Physical adaptations might serve to reduce adhesion to sticky spider silk, and thus

I predicted that C. californicum differs qualitatively in morphological traits such as setal structure and cuticular extensions of the tarsi (e.g. tarsal plantulae) when compared to wasp species that do not exhibit web landing behavior, such as its close relative S. caementarium. The features of the tarsi are of particular relevance to C. californicum’s hunting behavior, as the tarsi are the primary parts of the wasp’s body that are in contact with silk when it lands in a web (Coville, 1976). Following this prediction, I hypothesized that C. californicum body surfaces adhere less strongly to spider capture silk compared to other wasp species that do not exhibit web landing behavior. My prediction that C. californicum differs in physical traits that confer anti-adhesive qualities leads to the hypothesis that these wasps should be able to extricate themselves from spider webs more easily than other species of wasps that do not actively fly into spider webs and, hypothetically, adhere to viscid spider silk more strongly than C. californicum.

3 Thus, I predicted that C. californicum should be able to escape entrapment within an orb web in less time than these other wasp species.

4 CHAPTER II

METHODS

Study Organisms

I collected S. caementarium nests from Crown Point Ecology Center (Summit

County, Ohio) and various farms from Summit and Wayne Counties from late May through mid-June, from which I obtained adult C. californicum and S. caementarium after they finished their development. In addition, I obtained adults of these species via active collection with an entomological net in these same locations, as well as Panzner

Wetland (Summit County, Ohio) and the University of Akron campus (Summit County,

Ohio) from June through September. I marked all of these wasps with drops of nontoxic, oil-based paint on the pronotum for individual identification. All S. caementarium and C. californicum used in this study were kept in Lee’s Medium Kritter Keepers (29.9 x 19.7 x

20.3 centimeters) provided with 1:1 honey:water solution as food. Each cage only held wasps of a single species, and no more than four individuals were placed in the same cage at a time.

5 In addition to the mud daubers, I collected Polistes fuscatus (:

Polistinae), germanica (Vespidae: Vespinae), quadrifasciatus

(: Bembicinae), and Therion spp. () from June through

September. I selected these species both because they were relatively abundant in north- eastern Ohio, and also that they provide a phylogenetically diverse set of species to which

C. californicum can be compared to (Johnson et al. 2013; Branstetter et al. 2017; Peters et al. 2017). I collected these species from the following locations within Summit County,

Ohio: O’Neil Woods Metro Park, Hamilton Hills Metro Park, Crown Point Ecology

Center, Panzner Wetland, Hale Farm and Village, Bath Nature Preserve, the University of

Akron campus, and various local farms. I also collected from local farms in Wayne

County, Ohio. I obtained these wasps via active collection with an entomological net. I marked P. fuscatus with paint in the same way as the mud daubers for individual identification in the behavioral trials these three species were used in (see below in 2.4

Escape Trials). I kept these wasps in Lee’s Medium Kritter Keepers (29.9 x 19.7 x 20.3 centimeters) in the same manner as described for the mud daubers.

I collected Larinioides cornutus (Araneidae) to produce orb webs and adhesive silk for escape trials and adhesion tests, respectively. These primarily nocturnal, orb weaving spiders are prey for both C. californicum and S. caementarium (Peckham &

Peckham, 1898). I collected these spiders from a bridge on the Cuyahoga River in Akron,

Ohio and from a dock in Bath Pond in the Bath Nature Preserve (Summit County, Ohio).

I collected thirty of these spiders at the ending of May and beginning of June, and occasionally collected more throughout summer whenever spiders would perish. I kept these spiders individually in frames (41.4 cm x 40.2 cm x 12.7 cm) with removable sides,

6 and the frames themselves were kept in a pavilion in the Bath Nature Preserve. This meant that the L. cornutus were exposed to natural temperature and humidity. However, I sprayed frames with water vapor daily in the late afternoon before orb construction began, in order to encourage web building by providing a humidity that was more similar to the locations that the spiders were collected from, as these places were always near sources of water. I fed these spiders a weekly diet of crickets.

Scanning Electron Microscopy

I used an FEI Quanta 200 Environmental Scanning Electron Microscope to observe the tarsi of C. californicum, S. caementarium, and P. fuscatus. I did this to determine if C. californicum had any qualitative morphological differences from the other two wasp species that could help C. californicum reduce or eliminate adhesion to viscid spider silk. I focused my observations on the tarsi because these are the primary parts of the body that contact silk when C. californicum lands in the web to predate (Coville,

1976). I selected S. caementarium and P. fuscatus to make comparisons to a closely related species and a more distantly related species, respectively. I paid particular attention to setal structure and cuticular extensions, as these traits may be particularly important in influencing the strength of adhesion to sticky spider silk (Briceño &

Eberhard, 2012). I obtained all wasps used in this procedure from the University of

Akron insect collection.

7 Adhesion Trials

I used an MTS Nano Bionix (now Agilent Technologies) to measure the strength of adhesion between viscid capture silk acquired from L. cornutus webs and various body parts collected from the wasp species listed in 2.1 Study Organisms. To prepare these surfaces, I killed a wasp by placing it in a freezer for ten minutes. Immediately after this,

I removed the wasp’s abdomen, right middle leg, and right hind leg. I then removed the tarsus and femur from each of these legs. Additionally, I would remove the right wing and adhere its ventral surface to construction paper with double-sided tape, and I then used a rectangular hole puncher to create a 2.8 mm x 7.2 mm wing surface sample. I photographed all of these collected body parts on millimeter graph paper using an

Olympus Q-Color 5 digital camera, so that I could measure the width of each of these parts using ImageJ. After this, I glued these parts to nails (the ventral side was glued for the abdomens, dorsal side for the leg segments, and the bare side of the construction paper for the wing cutouts), and mounted them on the force sensor of the MTS Nano

Bionix. Above this, I mounted a strand of L. cornutus capture silk held in a cardboard frame (details below), and oriented the frame so that the thread sample was perpendicular to the wasp surface. I controlled the ambient humidity of the trial through the use of a plastic chamber set up around the thread and wasp surface sample to maintain a relative humidity of around 50%, as variation in this factor can affect the silk’s glue and its adhesion (Vollrath & Tillinghast, 1991; Amarpuri et al. 2015).

In each trial, the 12.6 mm thread sample was lowered down onto the wasp surface at 100 µm/sec, and continued to press down on the surface until the Nano Bionix

8 measured a load of 20 μN, at which point the thread sample was held still for six seconds to allow the glue to spread. After this, the thread sample was retracted at 100 µm/sec, and the force of adhesion between the thread of surface was measured over time until the thread was pulled off of the surface. From this, the work done to pull the capture thread off of the surface was calculated. I measured each wasp surface’s adhesion three times.

For leg segments, the thread sample was translated forward (0.5 mm) and laterally (2 mm), so that contact occurred at previously untouched parts of both the leg segment and the thread. To minimize edge effects, I kept the point of contact on the thread at least three millimeters from the cardboard mount the thread was held in. For abdomens and wing samples, I replaced the thread entirely, but still translated the new thread forward

(0.5 mm), so that it contacted parts of the abdomen or wing that had not previously touched sticky spider silk. I averaged the three replicates to give a single value of work of adhesion for each individual body part. Sample sizes are: NChalybion = 12, NSceliphron = 12,

NBicyrtes = 11, NPolistes = 12, NVespula = 12, and NTherion = 10. There were six instances in which a body part was contaminated, resulting in the loss of a replicate. These reduced sample sizes are as follows: NBicyrtes-Mid Tarsus = 11, NBicyrtes-Hind Femur = 11, NPolistes-Mid Tarsus =

11, NPolistes-Hind Femur = 11, NVespula-Wing = 11, NTherion-Mid Femur = 9.

I employed the following methods in order to account for intra-web and inter-web differences in adhesion. In order to minimize any possible differences in adhesion within a single web, I only collected threads from the bottom part of the bottom three sectors of the orb. In order to account for possible differences in adhesion between webs such as size or spacing of glue droplets, I tested three threads from every web used in the adhesion tests on a glass substrate to give an index of “stickiness” for each web. To do

9 this, I used the procedure described above, but used a six-millimeter-wide glass slide in place of a wasp surface. I washed the glass slide with isopropyl alcohol, and then dried the slide with pressurized air before testing each web. I then computed an index for work of adhesion (Wi) as:

푎푣푒푟푎푔푒 푎푑ℎ푒푠𝑖표푛 표푓 푠𝑖푙푘 푡표 푏표푑푦 푝푎푟푡 푊𝑖 = 푎푣푒푟푎푔푒 푎푑ℎ푒푠𝑖표푛 표푓 푠𝑖푙푘 푡표 푔푙푎푠푠 to control for variation in stickiness of different webs. I analyzed both the original data and this index data in the same fashion and found that it changed very few comparisons.

All values are therefore reported as relative to adhesion on glass (Wi).

Because some body parts varied in size among species, I created another set of data that controlled for length of contact between thread and insect surface, in order to compare the relative adhesive differences between the materials of these parts between species. To do this, I took images of the thread pressing down on a wasp part at the 20

μN load using a Photron Fastcam SA4. In ImageJ, I traced the length of contact of the thread to the part, using the overall part width (which I had already measured using millimeter graph paper) as a reference, to measure the total length of thread touching the surface. I did this for three times each for each part of each species, using parts from different individuals each time. From these values, I calculated a relative work index

(Wr) as:

푤표푟푘 표푓푎푑ℎ푒푠𝑖표푛 푚푚 표푓 푐표푛푡푎푐푡 푤𝑖푡ℎ 𝑖푛푠푒푐푡 푊푟 = 푤표푟푘 표푓 푎푑ℎ푒푠𝑖표푛 푡표 푔푙푎푠푠 푚푚 표푓 푔푙푎푠푠 푠푢푏푠푡푟푎푡푒 푤𝑖푑푡ℎ

10 where the work of adhesion per mm of insect surface was normalized to the work of adhesion of the same web’s silk on the glass control surface per width of glass surface (6 mm). Like the whole part data, I analyzed this material adhesion data without this glass correction, and again found that its effect was small and only changed a few comparisons.

Escape Trials

I conducted escape trials to gauge how quickly wasps can free themselves from entanglement within an orb web, testing the prediction that C. californicum can more easily escape spider webs than species that do not hunt by landing in spider webs. I used

S. caementarium and P. fuscatus as comparison species, because S. caementarium is a closely related spider hunting wasp that does not employ aggressive mimicry while P. fuscatus is a distantly related generalist of terrestrial . The C. californicum used in these trials did not have nests to stock, depriving them of their primary motivation to hunt spiders, and thus mitigate the chances of the wasp engaging in predatory behavior which might have increased the time it spent in the web. I used L. cornutus orb webs for this procedure. I only used webs that were at least somewhat symmetrical (the hub needed to be somewhere within the middle 20% of the overall width of the web). I conducted these trials indoors within a screen mesh tent (3.6 m x 3.0 m x 2.2 m) to prevent wasps from escaping, and I lit the trials using a Fovitec S-900D LED panel. All

11 wasp species used in this procedure are diurnal, and so I only conducted these tests between 10:00 and 16:00.

I conducted escape trials in pairs by randomly selecting one C. californicum and either one S. caementarium or one P. fuscatus. This created two sets of escape trials, C. californicum-S. caementarium paired trials (n=21) and C. californicum-P. fuscatus paired trials (n=20). I then randomly selected which of the two paired wasps would be tested first, and randomly selected whether the wasp would be thrown to the left of the hub or the right. I placed each tested wasp into a cylindrical plastic vial (diameter 3.2 cm, length

7.3 cm), to alleviate possible unintended differences in the throw arising from the handling of different species. I videotaped each trial using three Sony DCR-HC42 camcorders in order to record the trial from different angles. From these recordings, I measured the period between the wasp entering the web and the wasp escaping it.

Following each trial, I monitored the participating wasps. Whenever I observed a wasp’s death within 24 hours of its escape trial, I excluded its data from analysis. I did this because wasps within 24 hours of death may have had a deteriorated body condition, and the exclusion of these wasps mitigates the potential effects of poor body condition on the results. Analysis incorporating these excluded trials found that it did not change the results.

12 Statistical Analysis

I compared the work index values of every combination of species for each body part using Mann-Whitney U tests. I analyzed the work index calculated from the whole part adhesion (Wi) and the relative work index adjusted for size and shape (Wr) separately. I controlled for familywise error with post hoc sequential Bonferroni tests. For the escape trials, I compared each pair of wasps thrown into the same web, analyzing one set of comparisons between C. californicum and S. caementarium and a second set between C. californicum and P. fuscatus. I analyzed the escape trial data using Wilcoxon signed ranks tests. I performed all analyses using JMP Pro 14 statistics software.

13 CHAPTER III

RESULTS

There were no qualitative differences in tarsal morphology between C. californicum and S. caementarium observed in scanning electron microscopy. Both species had tarsal plantulae located distally and medially on each tarsomere (see Figure

1A). I did not observe any other differences in cuticular extensions of the tarsi between these two species. All three species had simple, unbranched setae, which is typical for

Hymenoptera (see Figure 1B).

Broadly speaking, the adhesion tests found that B. quadrifasciatus and V. germanica adhered to capture silk strongly, while both species of mud dauber wasps were consistently lower in adhesive strength. There were no significant differences in the relative adhesion of wasp abdomens to spider silk (see Figure 2A, 2B). C. californicum mid femurs adhered less strongly to capture silk than mid femurs of all other species, both in terms of whole femurs and relative to femur width (see Figure 2C, 2D). Mid tarsi showed stronger adhesion for B. quadrifasciatus and V. germanica and weaker adhesion for the mud daubers and P. fuscatus (see Figure 2E, 2F). The species comparisons were

14 quite similar between whole tarsi and tarsi controlled for size. There were few adhesive differences among whole hind femurs, limited to S. caementarium adhering less strongly than the B. quadrifasciatus and P. fuscatus (see Figure 2G). The relative hind femur adhesion formed a range, with B. quadrifasciatus and P. fuscatus among the higher end and the mud daubers adhering less strongly again (see Figure 2H). For both whole hind tarsi and relative hind tarsi adhesion, B. quadrifasciatus and V. germanica adhered strongly, while all other wasps had weaker adhesion (see Figure 2I, 2J). Relative wing adhesion formed another range, with B. quadrifasciatus and V. germanica achieving strong adhesion and P. fuscatus and the mud daubers achieving weak adhesion (see

Figure 2K). The specific p-values of these comparisons are given in Tables 1-3. These comparisons are described in greater detail in the appendix (see Tables A1-A11).

The difference in entanglement escape times between C. californicum and S. caementarium was insignificant (see Table 4, Figure 3), as was also the case with C. californicum and P. fuscatus (see Table 4, Figure 4). Around 30% of C. californicum trials lasted longer than three seconds, as did 20% of S. caementarium trials and 70% of

P. fuscatus trials. While on the web, all individuals appeared to vigorously struggle free, and flew away from the web once they had extricated themselves. This suggests that these wasps were solely motivated to leave, and their time in the web was not extended by a predatory response towards the spider that built the web.

15 Figure 3.1. Scanning electron microscope images of C. californicum tarsi. Images adjusted for brightness and contrast for clarity. (A) Chalybion californicum fore tarsus, ventral surface. Arrows indicate tarsal plantulae. Sceliphron caementarium and Polistes fuscatus tarsi have this same form, though tarsal plantulae are absent in P. fuscatus. (B) C. californicum mid tarsus setae. Note the simple, unbranched structure. S. caementarium and Polistes fuscatus setae have this same form.

16 Table 3.1. P-values of comparisons of abdomen and wing adhesion. Gold shaded cells refer to abdomen comparisons, and blue shaded cells refer to wing comparisons. Cells shaded with bolder color contain significant comparisons. Values not in parentheses correspond to relative adhesion, and those in parentheses correspond to whole part adhesion. P-values found significant after a sequential Bonferroni test are bolded. Chalybion Sceliphron Bicyrtes Polistes Vespula Therion 0.6236 0.7818 0.977 0.8852 0.0229 Chalybion (0.931) (0.2549) (0.7075) (0.8399) (0.0111) 0.8055 0.7508 0.7728 0.0092 Sceliphron 02855 (0.1858) (0.665) (1) (0.0192) 0.9509 0.9755 0.0151 Bicyrtes 0.0003 0.0005 (0.6009) (0.3401) (0.0011) 0.977 0.0602 Polistes 0.5066 0.4882 0.0002 (0.5067) (0.0321) 0.0378 Vespula 0.0001 0.0001 0.6934 <0.0001 (0.0927)

Therion 0.0076 0.0295 0.1927 0.0062 0.0724

17 Table 3.2. P-values of comparisons of mid femur and mid tarsus adhesion. Gold shaded cells refer to mid femur comparisons, and blue shaded cells refer to mid tarsus comparisons. Cells shaded with bolder color contain significant comparisons. Values not in parentheses correspond to relative adhesion, and those in parentheses correspond to whole part adhesion. P-values found significant after a sequential Bonferroni test are bolded. Chalybion Sceliphron Bicyrtes Polistes Vespula Therion 0.0002 <0.0001 <0.0001 <0.0001 0.0003 Chalybion (0.0015) (<0.0001) (0.0002) (<0.0001) (0.0032) 0.7508 0.0015 0.1748 0.2366 0.241 Sceliphron (0.0566) (0.0089) (0.2366) (0.1059) (0.5458) <0.0001 <0.0001 0.0525 0.0247 0.0303 Bicyrtes (<0.0001) (<0.0001) (0.1858) (0.3099) (0.0078) 0.0605 0.1166 0.0002 0.8852 0.644 Polistes (0.0525) (0.2549) (0.0005) (0.7075) (0.0597) 0.0004 0.0006 0.1062 0.0089 0.9717 Vespula (0.0004) (0.0014) (0.4483) (0.0089) (0.0252) 0.339 0.5752 0.0004 0.5495 0.0062 Therion (0.9212) (0.2225) (0.0004) (0.2048) (0.0027)

18 Table 3. P-values of comparisons of hind femur and hind tarsus adhesion. Gold shaded cells refer to hind femur comparisons, and blue shaded cells refer to hind tarsus comparisons. Cells shaded with bolder color contain significant comparisons. Values not in parentheses correspond to relative adhesion, and those in parentheses correspond to whole part adhesion. P-values found significant after a sequential Bonferroni test are bolded. Chalybion Sceliphron Bicyrtes Polistes Vespula Therion 1 0.0001 0.0011 0.0351 0.0004 Chalybion (0.1572) (0.0111) (0.0965) (0.1939) (0.1468) 0.686 0.0002 0.0019 0.0226 0.0007 Sceliphron (0.7075) (0.0007) (0.0035) (0.0688) (0.0076) <0.0001 <0.0001 0.0528 0.0518 0.089 Bicyrtes (<0.0001) (<0.0001) (0.098) (0.2485) (0.064) 0.3408 0.126 <0.0001 0.902 1 Polistes (0.0605) (0.1749) (0.0001) (0.8294) (0.7781) <0.0001 <0.0001 0.0905 0.0002 0.8431 Vespula (<0.0001) (<0.0001) (0.021) (0.0007) (0.6682) 0.2485 0.0927 0.0001 0.9474 0.0001 Therion (0.3068) (0.4887) (0.0001) (0.5752) (0.0001)

19 20 Figure 3.2. Comparisons of adhesion of various body parts between species. Work index indicates work done to pull a body part off of spider capture silk relative to work done to pull a glass side off of capture silk. Left column shows relative adhesion, right column shows whole part adhesion. The following outliers are not shown: (A): Bicyrtes – 30.44; (B): Bicyrtes – 17.03; (C): Bicyrtes – 82.46; (E): Bicyrtes – 122.44, Vespula – 118.99; (J): Bicyrtes – 3.94, Bicyrtes – 3.65; (K): Therion – 60.65. Letters indicate significant differences.

21 Table 3.4. Paired comparisons of time to escape entanglement of wasps thrown into orb webs.

Comparison Median Time 1 Median Time 2 n Z Prob. Chalybion- M = 2.62 sec M = 1.2 sec 21 1.27 0.1587 Sceliphron Chalybion Sceliphron Chalybion- M = 1.8 sec M = 3.78 sec 20 -1.57 0.1352 Polistes Chalybion Polistes

Figure 3.3. Comparisons of time to escape entanglement in an orb web between pairs of Chalybion californicum (Cc) and Sceliphron caementarium (Sc) thrown into the web. Each dot represents a single pair of wasps. Trials in which the S. caementarium took longer to escape have positive “Difference in Escape Time” values, while those in which the C. californicum took longer have negative values. The mean escape time of each pair is included to help visualize outliers.

22 Figure 3.4. Comparisons of time to escape entanglement in an orb web between pairs of Chalybion californicum (Cc) and Polistes fuscatus (Pf) thrown into the web. Each dot represents a single pair of wasps. Trials in which the P. fuscatus took longer to escape have positive “Difference in Escape Time” values, while those in which the C. californicum took longer have negative values. The mean escape time of each pair is included to help visualize outliers.

23 CHAPTER IV

DISCUSSION

I tested the hypothesis that the spider hunting wasp C. californicum possessed traits that prevented it from sticking to spider webs, thereby facilitating the wasp’s ability to mimic prey in webs while hunting. By comparing C. californicum to its fellow mud dauber S. caementarium and a variety of other wasp species, I expected to find that these other species would stick more strongly to capture silk than C. californicum. There was significant variation in how strongly surfaces from various wasp species adhered to capture silk, indicating that there are features and qualities of these insects that influence how strongly they stick to spider silk. Of the tested species, the mud daubers were consistently among the weakest adhering species, which is notable given that these two species are the only two spider specialists of the group examined here, though they also tended to perform similarly to the non-spider specialist P. fuscatus. Between each other,

C. californicum and S. caementarium generally adhered to silk about as strongly as each other, with the sole exception being the mid femurs. The similarity of tarsal adherence between these species is made less surprising by the similarity of their tarsal

24 morphologies (see Figure 1), though the wings and abdomens performed about the same between these species too. That these species performed so similarly even after controlling for variation in size of the tested surfaces suggests that any differences in the epicuticular chemistries of these two species did not meaningfully influence the strength of adhesion to capture silk. Consequently, it appears that C. californicum, despite its behavior of active entry into spider webs, has not evolved traits that reduce its adhesion beyond that of close relatives in order to predate in this manner.

Another hypothesis I tested was that C. californicum would be able to escape entanglement within an orb web more quickly than S. caementarium and P. fuscatus, as only C. californicum specializes in entering these webs. I found C. californicum’s two second median escape time was insignificantly lower than P. fuscatus’s four second median time, and insignificantly higher than S. caementarium’s one second median time

(see Table 4). However, another important perspective on this data is the distribution of escape times. The longer an insect remains within a spider web, the more time the spider has to recognize its presence and approach it. Escaping within the first three seconds of web contact should make an encounter with the spider unlikely, but entanglement beyond this makes this threat possible (Eberhard, 1989; Blackledge & Zevenbergen, 2006). Only

30% of C. californicum trials and 20% of S. caementarium trials had these wasps stuck in the web for more than three seconds while 70% of P. fuscatus were entangled for this period, indicating that entry into an orb web may be more dangerous for this species than it is for the mud daubers. It appears then that while C. californicum is not be uniquely well suited for escaping entanglement, it and its close relative S. caementarium are potentially more adept at it than unrelated, non-spider specialist wasps.

25 If the mud dauber species are indeed better adapted for escaping ensnarement within a web than unrelated species like P. fuscatus, how might they achieve this?

Though these species are largely similar in size, differences in body proportions could result in varying numbers of contacted threads, such as longer wings being at risk of touching more threads than shorter wings. Behavior in the web may also play a role here, as this has been found to vary between insect species (Suter, 1978; Nentwig, 1982).

Behaviors that simply work to pull a body part off of capture silk may result in the insect tumbling down the orb and adhering to more threads, increasing retention time (Olive,

1980; Eberhard, 1989; Blackledge & Zevenbergen, 2006). In contrast, behaviors utilizing wings to move perpendicularly to the orb could avoid this problem and escape entanglement more quickly, and some in-web behaviors like certain leg sweeping movements appear to work specifically to free the wings of threads (Suter, 1978; Olive,

1980; Blackledge & Zevenbergen, 2006). If mud dauber behavior in the web is more directed towards freeing their wings and using them to move perpendicularly to the web than P. fuscatus behavior is, this could result in the latter species taking longer to escape.

Determining whether the mud daubers truly are better adapted for escaping entanglement than other types of wasps will require expanding this research into more species however, as the limited number of species in this study would make this conclusion premature.

Regardless, that C. californicum was not the wasp with the lowest escape times suggests that it is not any more suited for escaping ensnarement than its close relative S. caementarium. Thus, it appears that C. californicum’s predatory strategy is not associated with unique physical anti-adhesion traits. So why doesn’t C. californicum adhere to the webs that it invades while hunting?

26

When C. californicum is searching its environment for spider webs, it flies with its legs stretched outward in front of it (Coville, 1976). As a result, when one of these wasps makes contact with a web and lands within it, only the wasp’s tarsi are in contact with the silk (Coville, 1976). Behaviorally it is simply minimizing contact with the adhesive silk in the web in much the same way that spiders “tip toe” through their webs

(Vollrath & Tillinghast, 1991). There are also occasional instances in which C. californicum becomes entangled within a web and cannot escape quickly enough to avoid by the spider it was hunting (Rau & Rau, 1918; Obin, 1982). Intriguingly, Obin

(1982) reported that wasps that “blundered” into a web (e.g. veered off of regular flight path due to an agonistic encounter) were more likely to become entangled, supporting the hypothesis that awareness of the web and the behavior of how the wasp lands in it plays a larger role than any physical anti-adhesion trait (Obin, 1982). In addition to this, research on other predators of web-building spiders suggests that they too may lack physical anti- adhesion traits. Assassin bugs stalking spiders within the web have been found to adhere to capture silk and can become entangled (Soley & Taylor, 2012; Soley & Taylor, 2013).

Winged insects that are otherwise vulnerable to ensnarement within a spider web have been found to have a much easier time escaping when only their legs are in contact with the silk, as this leaves their wings free to pull them off via flight (Olive, 1980; Blackledge

& Zevenbergen, 2006). This research supports the notion that physical anti-adhesion is not necessarily required in order to evolve a specialization on web-building spiders, and that the threat of becoming entangled may be sufficiently reduced by behavioral traits.

Though S. caementarium does not appear physically excluded from utilizing C. californicum’s hunting behavior, it has never been reported to do so (Eberhard, 1970;

27

Blackledge & Pickett, 2000). This may seem strange when considering that adopting this strategy could grant S. caementarium access to spiders that build three-dimensional webs, which may be more abundant than the spiders that this wasp does predate (Blackledge et al. 2003; Uma & Weiss, 2012). However, S. caementarium does capture cursorial spiders, which is a resource that is largely unexploited by C. californicum (Muma & Jeffers,

1945; Blackledge et al. 2003). These prey exclusivities seem consistent with the wasps’ hunting behaviors: C. californicum can lure out spiders that are well protected in three dimensional webs whereas S. caementarium is not dependent on a web’s threads and is thus well-suited for attacking spiders on the ground. The reason why C. californicum and

S. caementarium evolved these different strategies is perhaps related to C. californicum’s nesting habits, as it reuses old S. caementarium nests in lieu of building its own (Rau,

1928). C. californicum is thus an obligate cohabiter with its competitor S. caementarium.

Under the conditions of interspecific competition, selection may favor niche partitioning and specialization (Schoener, 1982; Pulliam, 1986; Futuyma & Morena, 1988; Wcislo,

1989; Ferry-Graham et al. 2002). If the hunting methods of these wasps determine what varieties of spider are available to them, selection could favor the reduction of interspecific competition by the differentiation of these behaviors.

C. californicum’s web landing behavior may thus represent an example of behavioral evolution due to extrinsic, ecological factors. While the physical traits of an organism determine what behaviors are possible for that species and which resources it may have access to, they are not the sole determinant of what particular niche is realized using those traits (Tinbergen, 1963; Futuyma & Morena, 1988; Schluter, 1996; Ferry-

Graham et al. 2002). The evolution of specialized behavioral traits is not necessarily

28 dependent on associated morphological or physiological specialization (Futuyma &

Morena, 1988). The observation of two species of predators hunting the same species of prey in substantially different manners, such as the case with C. californicum and S. caementarium, need not be the result of physical differences between the predators

(Futuyma & Morena, 1988; Wcislo, 1989; York & Fernald, 2017). It may instead be that they have been selected to behave differently by their ecologies, and that both behaviors are sufficiently well suited to predate that particular species of prey.

29

LITERATURE CITED

Amarpuri, G., C. Zhang, C. Diaz, B. D. Opell, T. A. Blackledge, A. Dhinojwala. 2015. Spiders Tune Glue Viscosity to Maximize Adhesion. ACS Nano 9(11): 11472- 11478.

Betz, O. & G. Kölsch. 2004. The role of adhesion in prey capture and predator defence in arthropods. Structure & Development 33: 3–30.

Blackledge, T. A., J. A. Coddington, & R. G. Gillespie. 2003. Are three-dimensional spider webs defensive adaptations? Ecology Letters 6: 13-18.

Blackledge, T. A. & K. M. Pickett. 2000. Predatory Interactions between Mud-Dauber Wasps (, Sphecidae) and Argiope (Araneae, Araneidae) in Captivity. The Journal of Arachnology 28(2): 211-216.

Blackledge, T. A. & J. M. Zevenbergen. 2006. Mesh Width Influences Prey Retention in Spider Orb Webs. Ethology 112: 1194-1201.

Bohart, R. M. & A. S. Menke. 1976. Sphecid Wasps of the World: A Generic Revision. University of California Press, Berkeley, .

Branstetter, M. G., B. N. Danforth, J. P. Pitts, B. C. Faircloth, P. S. Ward, M. L. Buffington, M. W. Gates, R. R. Kula, & S. G. Brady. 2017. Phylogenomic Insights into the Evolution of Stinging Wasps and the Origins of and . Current Biology 27(7): 1019-1025.

30 Briceño, R. D. & W. G. Eberhard. 2012. Spiders avoid sticking to their webs: clever leg movements, branched drip-tip setae, and anti-adhesive surface. Naturwissenschaften 99: 337–341.

Coville, R. E. 1976. Predatory behavior of the , Chalybion californicum (Hymenoptera, Sphecidae). Pan-Pacific Entomologist 52(3): 229-233.

Eberhard, W. 1970. The predatory behavior of two wasps, Agenoideus humilis (Pompilidae) and Sceliphron caementarium (Sphecidae), on the orb weaving spider Araneus cornutus (Araneidae). Psyche 77(2): 243-251.

Eberhard, W. G. 1989. Effects of orb web orientation and spider size on prey retention. Bulletin of the British Arachnological Society 8: 45-48.

Ferry-Graham, L. A., D. I. Bolnick, & P. C. Wainwright. 2002. Using Functional Morphology to Examine the Ecology and Evolution of Specialization. Integrative and Comparative Biology 42: 265-277.

Futuyma, D. J. & G. Moreno. 1988. The Evolution of Ecological Specialization. Annual Review of Ecology and Systematics 19: 207-233.

Horner, N. V. & J. H. Klein. 1979. Spider Prey of Two Mud Dauber Wasp Species in Comanche County, Oklahoma (Hymenoptera: Sphecidae). Environmental Entomology 8(1): 30-31.

Jeanne, R. L. 1972. Social biology of the neotropical wasp Mischocyttarus drewseni. Bulletin of the Museum of Comparative Zoology of Harvard 144(3): 63-150.

Johnson, B. R., M. L. Boroweic, J. C. Chiu, E. K. Lee, J. Atallah, & P. S. Ward. 2013. Phylogenomics resolves evolutionary relationships among ants, bees, and wasps. Current Biology 23(20): 2058-2062.

31

Kropf, C., D. Bauer, T. Schläppi, & A. Jacob. 2012. An organic coating keeps orb- weaving spiders (Araneae, Araneoidea, Araneidae) from sticking to their own capture threads. Journal of Zoological Systematics and Evolutionary Research 50(1): 14-18.

Muma, M. H. & W. F. Jeffers. 1945. Studies of the Spider Prey of Several Mud-Dauber Wasps. Annals of the Entomological Society of America 38(2): 245-255.

Nentwig, W. 1982. Why Do Only Certain Insects Escape from a Spider's Web? Oecologia 53: 412-417.

Obin, M. S. 1982. Spiders living at wasp nesting sites: what constrains predation by mud- daubers? Psyche 89: 321-336.

Olive, C. W. 1980. Foraging Specialization in Orb-Weaving Spiders. Ecology 61: 1133- 1144.

Peckham, G. W. & E. G. Peckham. 1898. On the Instincts and Habits of the Solitary Wasps. Wisconsin Geological and Natural History Survey Bulletin 2: 1-245.

Peters, R. S., L. Krogmann, C. Mayer, A. Donath, S. Gunkel, K. Meusermann, A. Kozlov, L. Podsiadlowski, M. Petersen, R. Lanfear, P. A. Diez, J. Heraty, K. M. Kjer, S. Klopfstein, R. Meier, C. Polidori, T. Schmitt, S. Liu, X. Zhou, T. Wappler, J. Rust, B. Misof, & O. Niehuis. 2017. Evolutionary History of the Hymenoptera. Current Biology 27: 1013-1018.

Pulliam, H. R. 1986. Niche Expansion and Contraction in a Variable Environment. American Zoologist 26: 71-79.

Rau, P. 1928. The Nesting Habits of the Wasp, Chalybion caeruleum. Annals of the Entomological Society of America 21(1): 25-35.

32 Rau, P. & N. Rau. 1918. Wasp studies afield. Princeton University Press, Princeton, United States.

Schluter, D. 1996. Ecological Causes of Adaptive Radiation. American Naturalist 148: S40-S64.

Schoener, T. W. 1982. The Controversy over Interspecific Competition: Despite spirited criticism, competition continues to occupy a major domain in ecological thought. American Scientist 70: 586-595.

Soley, F. G. & P. W. Taylor. 2012. Araneophagic assassin bugs choose routes that minimize risk of detection by web-building spiders. Animal Behaviour 84: 315- 321.

Soley, F. G. & P. W. Taylor. 2013. Ploys and counterploys of assassin bugs and their dangerous spider prey. Behaviour 150: 397-425.

Suter, R. B. 1978. Cyclosa turbinata (Araneae, Araneidae): Prey Discrimination via Web-Borne Vibrations. Behavioral Ecology and Sociobiology 3(3): 283-296.

Tinbergen, N. 1963. On Aims and Methods of Ethology. Zeitschrift für Tierpsychologie 20: 410–433.

Uma, D. B. & M. R. Weiss. 2012. Flee or Fight: Ontogenetic Changes in the Behavior of Cobweb Spiders in Encounters With Spider-Hunting Wasps. Environmental Entomology 41(6): 1474-1480.

Vollrath, F. & E. K. Tillinghast. 1991. Glycoprotein Glue Beneath a Spider Web's Aqueous Coat. Naturwissenschaften 78: 557- 559.

Wcislo, W. T. 1989. Behavioral Environments and Evolutionary Change. Annual Review of Ecology and Systematics 20: 137-169.

33 York, R. A. & R. D. Fernald. 2017. The Repeated Evolution of Behavior. Frontiers in Ecology and Evolution 4(143): doi: 10.3389/fevo.2016.00143.

York, R. A., C. Patil, C. D. Hulsey, O. Anoruo, J. T. Streelman, & R. D. Fernald. 2015. Evolution of bower building in Lake Malawi cichlid fish: phylogeny, morphology, and behavior. Frontiers in Ecology and Evolution 3(18): doi: 10.3389/fevo.2015.00018.

34

APPENDIX

Table A1. Comparisons of abdomen relative adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass, corrected for length of contact between silk and the tested surface. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.0124 MChalybion = 0.0138 71 0.7818 Chalybion n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0124 MPolistes = 0.0178 67.5 0.9509 Polistes n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0124 MSceliphron = 0.0129 70.5 0.8055 Sceliphron n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0124 MTherion = 0.0329 90 0.0151 Therion n = 11 n = 10 Bicyrtes- MBicyrtes = 0.0124 MVespula = 0.017 67 0.9755 Vespula n = 11 n = 12 Chalybion- MChalybion = 0.0138 MPolistes = 0.0178 73 0.977 Polistes n = 12 n = 12 Chalybion- MChalybion = 0.0138 MSceliphron = 0.0129 81 0.6236 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.0138 MTherion = 0.0329 95 0.0229 Therion n = 12 n = 10 Chalybion- MChalybion = 0.0138 MVespula = 0.017 75 0.8852 Vespula n = 12 n = 12 Polistes- MPolistes = 0.0178 MSceliphron = 0.0129 78 0.7508 Sceliphron n = 12 n = 12 Polistes- MPolistes = 0.0178 MTherion = 0.0329 89 0.0602 Therion n = 12 n = 10 Polistes- MPolistes = 0.0178 MVespula = 0.017 73 0.977 Vespula n = 12 n = 12 Sceliphron- MSceliphron = 0.0129 MTherion = 0.0329 100 0.0092 Therion n = 12 n = 10 Sceliphron- MSceliphron = 0.0129 MVespula = 0.017 77.5 0.7728 Vespula n = 12 n = 12 Therion- MTherion = 0.0329 MVespula = 0.017 92 0.0378 Vespula n = 10 n = 12

35 Table A2. Comparisons of abdomen whole part adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.00785 MChalybion = 0.00612 85 0.2549 Chalybion n = 11 n = 12 Bicyrtes- MBicyrtes = 0.00785 MPolistes = 0.00899 75 0.6009 Polistes n = 11 n = 12 Bicyrtes- MBicyrtes = 0.00785 MSceliphron = 0.00609 88 0.1858 Sceliphron n = 11 n = 12 Bicyrtes- MBicyrtes = 0.00785 MTherion = 0.00247 102 0.0011 Therion n = 11 n = 10 Bicyrtes- MBicyrtes = 0.00785 MVespula = 0.00641 82 0.3401 Vespula n = 11 n = 12 Chalybion- MChalybion = 0.00612 MPolistes = 0.00899 79 0.7075 Polistes n = 12 n = 12 Chalybion- MChalybion = 0.00612 MSceliphron = 0.00609 74 0.931 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.00612 MTherion = 0.00247 99 0.0111 Therion n = 12 n = 10 Chalybion- MChalybion = 0.00612 MVespula = 0.00641 76 0.8399 Vespula n = 12 n = 12 Polistes- MPolistes = 0.00899 MSceliphron = 0.00609 80 0.665 Sceliphron n = 12 n = 12 Polistes- MPolistes = 0.00899 MTherion = 0.00247 93 0.0321 Therion n = 12 n = 10 Polistes- MPolistes = 0.00899 MVespula = 0.00641 84 0.5067 Vespula n = 12 n = 12 Sceliphron- MSceliphron = 0.00609 MTherion = 0.00247 96 0.0192 Therion n = 12 n = 10 Sceliphron- MSceliphron = 0.00609 MVespula = 0.00641 72 1 Vespula n = 12 n = 12 Therion- MTherion = 0.00247 MVespula = 0.00641 86 0.0927 Vespula n = 10 n = 12

36 Table A3. Comparisons of mid femur relative adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass, corrected for length of contact between silk and the tested surface. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.136 MChalybion = 0.0103 132 <0.0001 Chalybion n = 11 n = 12 Bicyrtes- MBicyrtes = 0.136 MPolistes = 0.0673 98 0.0525 Polistes n = 11 n = 12 Bicyrtes- MBicyrtes = 0.136 MSceliphron = 0.0401 118 0.0015 Sceliphron n = 11 n = 12 Bicyrtes- MBicyrtes = 0.136 MTherion = 0.0635 78.5 0.0303 Therion n = 11 n = 9 Bicyrtes- MBicyrtes = 0.136 MVespula = 0.0652 103 0.0247 Vespula n = 11 n = 12 Chalybion- MChalybion = 0.0103 MPolistes = 0.0673 142 <0.0001 Polistes n = 12 n = 12 Chalybion- MChalybion = 0.0103 MSceliphron = 0.0401 136 0.0002 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.0103 MTherion = 0.0635 106 0.0003 Therion n = 12 n = 9 Chalybion- MChalybion = 0.0103 MVespula = 0.0652 141 <0.0001 Vespula n = 12 n = 12 Polistes- MPolistes = 0.0673 MSceliphron = 0.0401 96 0.1748 Sceliphron n = 12 n = 12 Polistes- MPolistes = 0.0673 MTherion = 0.0635 61 0.644 Therion n = 12 n = 9 Polistes- MPolistes = 0.0673 MVespula = 0.0652 75 0.8852 Vespula n = 12 n = 12 Sceliphron- MSceliphron = 0.0401 MTherion = 0.0635 71 0.241 Therion n = 12 n = 9 Sceliphron- MSceliphron = 0.0401 MVespula = 0.0652 93 0.2366 Vespula n = 12 n = 12 Therion- MTherion = 0.0635 MVespula = 0.0652 55 0.9717 Vespula n = 9 n = 12

37 Table A4. Comparisons of mid femur whole part adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.0098 MChalybion = 0.00136 130 <0.0001 Chalybion n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0098 MPolistes = 0.00566 88 0.1858 Polistes n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0098 MSceliphron = 0.0039 109 0.0089 Sceliphron n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0098 MTherion = 0.00305 85 0.0078 Therion n = 11 n = 9 Bicyrtes- MBicyrtes = 0.0098 MVespula = 0.00759 83 0.3099 Vespula n = 11 n = 12 Chalybion- MChalybion = 0.00136 MPolistes = 0.00566 138 0.0002 Polistes n = 12 n = 12 Chalybion- MChalybion = 0.00136 MSceliphron = 0.0039 127.5 0.0015 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.00136 MTherion = 0.00305 96 0.0032 Therion n = 12 n = 9 Chalybion- MChalybion = 0.00136 MVespula = 0.00759 141 <0.0001 Vespula n = 12 n = 12 Polistes- MPolistes = 0.00566 MSceliphron = 0.0039 93 0.2366 Sceliphron n = 12 n = 12 Polistes- MPolistes = 0.00566 MTherion = 0.00305 81 0.0597 Therion n = 12 n = 9 Polistes- MPolistes = 0.00566 MVespula = 0.00759 79 0.7075 Vespula n = 12 n = 12 Sceliphron- MSceliphron = 0.0039 MTherion = 0.00305 63 0.5458 Therion n = 12 n = 9 Sceliphron- MSceliphron = 0.0039 MVespula = 0.00759 100.5 0.1059 Vespula n = 12 n = 12 Therion- MTherion = 0.00305 MVespula = 0.00759 86 0.0252 Vespula n = 9 n = 12

38 Table A5. Comparisons of mid tarsus relative adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass, corrected for length of contact between silk and the tested surface. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.482 MChalybion = 0.0382 120 <0.0001 Chalybion n = 10 n = 12 Bicyrtes- MBicyrtes = 0.482 MPolistes = 0.0695 108 0.0002 Polistes n = 10 n = 11 Bicyrtes- MBicyrtes = 0.482 MSceliphron = 0.0385 120 <0.0001 Sceliphron n = 10 n = 12 Bicyrtes- MBicyrtes = 0.482 MTherion = 0.0421 97 0.0004 Therion n = 10 n = 10 Bicyrtes- MBicyrtes = 0.482 MVespula = 0.232 85 0.1062 Vespula n = 10 n = 12 Chalybion- MChalybion = 0.0382 MPolistes = 0.0695 97 0.0605 Polistes n = 12 n = 11 Chalybion- MChalybion = 0.0382 MSceliphron = 0.0385 78 0.7508 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.0382 MTherion = 0.0421 75 0.339 Therion n = 12 n = 10 Chalybion- MChalybion = 0.0382 MVespula = 0.232 134 0.0004 Vespula n = 12 n = 12 Polistes- MPolistes = 0.0695 MSceliphron = 0.0385 92 0.1166 Sceliphron n = 11 n = 12 Polistes- MPolistes = 0.0695 MTherion = 0.0421 64 0.5495 Therion n = 11 n = 10 Polistes- MPolistes = 0.0695 MVespula = 0.232 109 0.0089 Vespula n = 11 n = 12 Sceliphron- MSceliphron = 0.0385 MTherion = 0.0421 69 0.5752 Therion n = 12 n = 10 Sceliphron- MSceliphron = 0.0385 MVespula = 0.232 132 0.0006 Vespula n = 12 n = 12 Therion- MTherion = 0.0421 MVespula = 0.232 102 0.0062 Vespula n = 10 n = 12

39 Table A6. Comparisons of mid tarsus whole part adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.0154 MChalybion = 0.00161 120 <0.0001 Chalybion n = 10 n = 12 Bicyrtes- MBicyrtes = 0.0154 MPolistes = 0.00312 105 0.0005 Polistes n = 10 n = 11 Bicyrtes- MBicyrtes = 0.0154 MSceliphron = 0.00222 120 <0.0001 Sceliphron n = 10 n = 12 Bicyrtes- MBicyrtes = 0.0154 MTherion = 0.00138 97 0.0004 Therion n = 10 n = 10 Bicyrtes- MBicyrtes = 0.0154 MVespula = 0.0103 72 0.4483 Vespula n = 10 n = 12 Chalybion- MChalybion = 0.00161 MPolistes = 0.00312 98 0.0525 Polistes n = 12 n = 11 Chalybion- MChalybion = 0.00161 MSceliphron = 0.00222 105.5 0.0566 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.00161 MTherion = 0.00138 62 0.9212 Therion n = 12 n = 10 Chalybion- MChalybion = 0.00161 MVespula = 0.0103 134 0.0004 Vespula n = 12 n = 12 Polistes- MPolistes = 0.00312 MSceliphron = 0.00222 85 0.2549 Sceliphron n = 11 n = 12 Polistes- MPolistes = 0.00312 MTherion = 0.00138 73.5 0.2048 Therion n = 11 n = 10 Polistes- MPolistes = 0.00312 MVespula = 0.0103 109 0.0089 Vespula n = 11 n = 12 Sceliphron- MSceliphron = 0.00222 MTherion = 0.00138 79 0.2225 Therion n = 12 n = 10 Sceliphron- MSceliphron = 0.00222 MVespula = 0.0103 128 0.0014 Vespula n = 12 n = 12 Therion- MTherion = 0.00138 MVespula = 0.0103 106 0.0027 Vespula n = 10 n = 12

40 Table A7. Comparisons of hind femur relative adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass, corrected for length of contact between silk and the tested surface. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.191 MChalybion = 0.0291 118 0.0001 Chalybion n = 10 n = 12 Bicyrtes- MBicyrtes = 0.191 MPolistes = 0.0752 83 0.0528 Polistes n = 10 n = 11 Bicyrtes- MBicyrtes = 0.191 MSceliphron = 0.0234 117 0.0002 Sceliphron n = 10 n = 12 Bicyrtes- MBicyrtes = 0.191 MTherion = 0.0789 73 0.089 Therion n = 10 n = 10 Bicyrtes- MBicyrtes = 0.191 MVespula = 0.0831 90 0.0518 Vespula n = 10 n = 12 Chalybion- MChalybion = 0.0291 MPolistes = 0.0752 119.5 0.0011 Polistes n = 12 n = 11 Chalybion- MChalybion = 0.0291 MSceliphron = 0.0234 72 1 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.0291 MTherion = 0.0789 114 0.0004 Therion n = 12 n = 10 Chalybion- MChalybion = 0.0291 MVespula = 0.0831 109 0.0351 Vespula n = 12 n = 12 Polistes- MPolistes = 0.0752 MSceliphron = 0.0234 117 0.0019 Sceliphron n = 11 n = 12 Polistes- MPolistes = 0.0752 MTherion = 0.0789 55.5 1 Therion n = 11 n = 10 Polistes- MPolistes = 0.0752 MVespula = 0.0831 68.5 0.902 Vespula n = 11 n = 12 Sceliphron- MSceliphron = 0.0234 MTherion = 0.0789 112 0.0007 Therion n = 12 n = 10 Sceliphron- MSceliphron = 0.0234 MVespula = 0.0831 112 0.0226 Vespula n = 12 n = 12 Therion- MTherion = 0.0789 MVespula = 0.0831 63.5 0.8431 Vespula n = 10 n = 12

41 Table A8. Comparisons of hind femur whole part adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.0113 MChalybion = 0.00371 99 0.0111 Chalybion n = 10 n = 12 Bicyrtes- MBicyrtes = 0.0113 MPolistes = 0.00509 79 0.098 Polistes n = 10 n = 11 Bicyrtes- MBicyrtes = 0.0113 MSceliphron = 0.00222 112 0.0007 Sceliphron n = 10 n = 12 Bicyrtes- MBicyrtes = 0.0113 MTherion = 0.00508 75 0.064 Therion n = 10 n = 10 Bicyrtes- MBicyrtes = 0.0113 MVespula = 0.00735 78 0.2485 Vespula n = 10 n = 12 Chalybion- MChalybion = 0.00371 MPolistes = 0.00509 93.5 0.0965 Polistes n = 12 n = 11 Chalybion- MChalybion = 0.00371 MSceliphron = 0.00222 97 0.1572 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.00371 MTherion = 0.00508 82.5 0.1468 Therion n = 12 n = 10 Chalybion- MChalybion = 0.00371 MVespula = 0.00735 95 0.1939 Vespula n = 12 n = 12 Polistes- MPolistes = 0.00509 MSceliphron = 0.00222 114 0.0035 Sceliphron n = 11 n = 12 Polistes- MPolistes = 0.00509 MTherion = 0.00508 59.5 0.7781 Therion n = 11 n = 10 Polistes- MPolistes = 0.00509 MVespula = 0.00735 70 0.8294 Vespula n = 11 n = 12 Sceliphron- MSceliphron = 0.00222 MTherion = 0.00508 101 0.0076 Therion n = 12 n = 10 Sceliphron- MSceliphron = 0.00222 MVespula = 0.00735 104 0.0688 Vespula n = 12 n = 12 Therion- MTherion = 0.00508 MVespula = 0.00735 67 0.6682 Vespula n = 10 n = 12

42 Table A9. Comparisons of hind tarsus relative adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass, corrected for length of contact between silk and the tested surface. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.368 MChalybion = 0.0344 131 <0.0001 Chalybion n = 11 n = 12 Bicyrtes- MBicyrtes = 0.368 MPolistes = 0.0465 130 <0.0001 Polistes n = 11 n = 12 Bicyrtes- MBicyrtes = 0.368 MSceliphron = 0.0287 132 <0.0001 Sceliphron n = 11 n = 12 Bicyrtes- MBicyrtes = 0.368 MTherion = 0.0453 110 0.0001 Therion n = 11 n = 10 Bicyrtes- MBicyrtes = 0.368 MVespula = 0.248 94 0.0905 Vespula n = 11 n = 12 Chalybion- MChalybion = 0.0344 MPolistes = 0.0465 89 0.3408 Polistes n = 12 n = 12 Chalybion- MChalybion = 0.0344 MSceliphron = 0.0287 79.5 0.686 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.0344 MTherion = 0.0453 78 0.2485 Therion n = 12 n = 10 Chalybion- MChalybion = 0.0344 MVespula = 0.248 143 <0.0001 Vespula n = 12 n = 12 Polistes- MPolistes = 0.0465 MSceliphron = 0.0287 99 0.126 Sceliphron n = 12 n = 12 Polistes- MPolistes = 0.0465 MTherion = 0.0453 61.5 0.9474 Therion n = 12 n = 10 Polistes- MPolistes = 0.0465 MVespula = 0.248 137 0.0002 Vespula n = 12 n = 12 Sceliphron- MSceliphron = 0.0287 MTherion = 0.0453 86 0.0927 Therion n = 12 n = 10 Sceliphron- MSceliphron = 0.0287 MVespula = 0.248 144 <0.0001 Vespula n = 12 n = 12 Therion- MTherion = 0.0453 MVespula = 0.248 118 0.0001 Vespula n = 10 n = 12

43 Table A10. Comparisons of hind tarsus whole part adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.0181 MChalybion = 0.00159 132 <0.0001 Chalybion n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0181 MPolistes = 0.0026 129 0.0001 Polistes n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0181 MSceliphron = 0.0018 132 <0.0001 Sceliphron n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0181 MTherion = 0.00206 110 0.0001 Therion n = 11 n = 10 Bicyrtes- MBicyrtes = 0.0181 MVespula = 0.00922 104 0.021 Vespula n = 11 n = 12 Chalybion- MChalybion = 0.00159 MPolistes = 0.0026 105 0.0605 Polistes n = 12 n = 12 Chalybion- MChalybion = 0.00159 MSceliphron = 0.0018 79 0.7075 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.00159 MTherion = 0.00206 76 0.3068 Therion n = 12 n = 10 Chalybion- MChalybion = 0.00159 MVespula = 0.00922 142 <0.0001 Vespula n = 12 n = 12 Polistes- MPolistes = 0.0026 MSceliphron = 0.0018 96 0.1749 Sceliphron n = 12 n = 12 Polistes- MPolistes = 0.0026 MTherion = 0.00206 69 0.5752 Therion n = 12 n = 10 Polistes- MPolistes = 0.0026 MVespula = 0.00922 131 0.0007 Vespula n = 12 n = 12 Sceliphron- MSceliphron = 0.0018 MTherion = 0.00206 71 0.4887 Therion n = 12 n = 10 Sceliphron- MSceliphron = 0.0018 MVespula = 0.00922 143 <0.0001 Vespula n = 12 n = 12 Therion- MTherion = 0.00206 MVespula = 0.00922 118 0.0001 Vespula n = 10 n = 12

44 Table A11. Comparisons of wing relative adhesion. The median value is a work index calculated by dividing the adhesive work of silk on wasp body part by adhesive work of silk on glass, corrected for length of contact between silk and the tested surface. P-values found significant after a sequential Bonferroni test are bolded.

Comparison Median 1 Median 2 U Value Prob. (n) (n) Bicyrtes- MBicyrtes = 0.0898 MChalybion = 0.0123 125 0.0003 Chalybion n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0898 MPolistes = 0.0163 127 0.0002 Polistes n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0898 MSceliphron = 0.0179 123 0.0005 Sceliphron n = 11 n = 12 Bicyrtes- MBicyrtes = 0.0898 MTherion = 0.0604 74 0.1927 Therion n = 11 n = 10 Bicyrtes- MBicyrtes = 0.0898 MVespula = 0.11 67 0.6934 Vespula n = 11 n = 11 Chalybion- MChalybion = 0.0123 MPolistes = 0.0163 84 0.5066 Polistes n = 12 n = 12 Chalybion- MChalybion = 0.0123 MSceliphron = 0.0179 91 0.2855 Sceliphron n = 12 n = 12 Chalybion- MChalybion = 0.0123 MTherion = 0.0604 101 0.0076 Therion n = 12 n = 10 Chalybion- MChalybion = 0.0123 MVespula = 0.11 129 0.0001 Vespula n = 12 n = 11 Polistes- MPolistes = 0.0163 MSceliphron = 0.0179 84.5 0.4882 Sceliphron n = 12 n = 12 Polistes- MPolistes = 0.0163 MTherion = 0.0604 102 0.0062 Therion n = 12 n = 10 Polistes- MPolistes = 0.0163 MVespula = 0.11 120 <0.0001 Vespula n = 12 n = 11 Sceliphron- MSceliphron = 0.0179 MTherion = 0.0604 93.5 0.0295 Therion n = 12 n = 10 Sceliphron- MSceliphron = 0.0179 MVespula = 0.11 129 0.0001 Vespula n = 12 n = 11 Therion- MTherion = 0.0604 MVespula = 0.11 81 0.0724 Vespula n = 10 n = 11

45