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Author: Phangurha, Josh Title: Construction of the orb web in constant and changing abiotic conditions

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Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. Construction of the orb web in constant and changing abiotic conditions

Joshua Singh Phangurha

A dissertation submitted to the University of Bristol in the accordance with the requirements for award of the degree of MSc by Research in the Faculty of Science.

School of Biological Sciences

Date of submission: 31/08/2018

Word count: 15,060 Abstract

Orb weaving spiders are thought to alter their web-building in different abiotic conditions to maximise prey capture success, while being as silk-efficient as possible. However, there is limited evidence to support this, along with a lack of focus on web variation over time under constant conditions. It is important to better understand web-building in constant and changing abitoic environments to determine if spiders perform adaptive manipulations of their webs. Here, both of these concepts are investigated and the hypothesis that web construction under constant abiotic conditions will not change over time, due to a lack of environmental cues for spiders to respond to, is tested. It is also predicted that Zygiella x- notata will reduce the prey capture aspects in high light intensities, when prey would be more active, and silk can be preserved. The webs of both species were monitored in a constant abiotic environment and Z. x-notata web building behaviour was compared in varying environmental light intensities. These experiments explored patterns of variation in the web characteristics of Argiope bruennichi and Zygiella x-notata over time in a constant abiotic environment and identified significant differences in the web building of Z. x-notata in bright and dark conditions. Results show an overall lack of variation in the webs of A. bruennichi over time apart from radii number in webs of adult females and upper mesh spacing in juvenile webs, and no significant variation in the webs of Z. x-notata over time. No significant differences in the web geometry of Z. x-notata webs occurred in the light intensity treatment, except for lower mesh height becoming reduced in darker conditions. These findings suggest that varying abiotic factors, overall, do not influence the adaptive web building decisions of an orb weaver and could only marginally impact specific web aspects. Dedication and acknowledgments

I would like to thank Beth Mortimer and Daniel Robert for supervising this project in the Life Sciences building, Paul Chappell and his colleagues at the chemistry department for building the frame set up and the British Ecological Society and Bristol Alumni foundation for funding my travel to Mexico to share this research at the American Arachnological Society’s annual meeting.

All photographs used in this thesis were taken by the author. Image 1.1 (2.2MB), 1.2 (8.69MB), 1.4 (467KB), 2.1 (5.89MB), 2.2 (6.35MB), 2.3 (6.58MB), 2.4 (7.26MB), 4.1 (337KB), 4.2 (1.11MB), 4.3 (306KB) and 4.4 (435KB) were all edited in Picasa®. Image 1.3 (9MB) was downloaded from Leica® microscopy software.

Author’s declaration

I declare that the work in this dissertation was carried out in accordance with the requirements of the University's Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate's own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author.

SIGNED: ...... DATE:......

Table of contents

Chapter 1 – Introduction and background ………………………………………………...... 1 1.1 – Background on foraging method variation in the natural world ………………………………… 1 1.2 – Biotic influence on web construction ………………………………………………………………………… 2 1.3 – Spider condition ……………………………………………………………………………………………………….. 3 1.4 – Web building influenced by predators …………………………………………………………………………………… 3

1.5 – Wind ……………………………………………………………………………………………………………………………………... 4

1.6 – Light ………………………………………………………………………………………………………………………………………. 5

1.7 – Temperature …………………………………………………………………………………………………………………………. 6

1.8 – Humidity ……………………………………………………………………………………………………………………………….. 7

1.9 – Intraspecific web variation ……………………………………………………………………………………………………. 8

1.10 – Other factors affecting web building ………………………………………………………………………………….. 10

1.11 – Stabilimenta – a prey attractant? ……………………………………………………………………………………….. 11

1.12 – Web damage prevention function of stabilimenta ……………………………………………………………… 12

1.13 – Stabilimenta for predator avoidance ………………………………………………………………………………….. 13

1.14 – Unexplained stabilimenta variation ……………………………………………………………………………………. 14

1.15 – Aim of study ………………………………………………………………………………………………………………………. 14

1.16 – Study species’ ……………………………………………………………………………………………………………………. 15

Chapter 2 – Methodology …………………………………………………………………………………………………. 18 2.1 – Argiope bruennichi and Zygiella x-notata collection ……………………………………………………………... 18

2.2 – Maintenance ………………………………………………………………………………………………………………………… 18

2.3 – Morphometrics ……………………………………………………………………………………………………………………. 19

2.4 – Environmental conditions …………………………………………………………………………………………………….. 21

2.5 – Light intensity experiment (Zygiella x-notata) ………………………………………………………………………. 22

2.6 – Web measurements …………………………………………………………………………………………………………….. 22

2.7 – Statistical methods ………………………………………………………………………………………………………………. 26

Chapter 3 – Results ……………………………………………………………………………………………………………… 26 3.1 – Web building variation under constant abiotic conditions …………………………………………………… 27

3.2 – Juvenile Argiope bruennichi …………………………………………………………………………………………………. 27

3.3 – Adult Argiope bruennichi …………………………………………………………………………………………………….. 34

3.4 – Stabilimenta building ………………………………………………………………………………………………………….. 40

3.5 – Zygiella x-notata …………………………………………………………………………………………………………………. 40

3.6 – Light intensity experiment ………………………………………………………………………………………………….. 47

Chapter 4 – Discussion ……………………………………………………………………………………………………… 49 4.1 – Juvenile upper mesh variation and overall mesh consistency …………………………………………….. 50

4.2 – Spatial constraints ………………………………………………………………………………………………………………. 52

4.3 – Web building experience ……………………………………………………………………………………………………. 54

4.4 – Size limitation hypothesis …………………………………………………………………………………………………… 54

4.5 – Adult Argiope bruennichi radii variation …………………………………………………………………………….. 55

4.6 – Biotic factors more influential on web construction? …………………………………………………………. 56

4.6.1 – Effect of food intake ……………………………………………………………………………………………….. 56

4.6.2 – Prey type …………………………………………………………………………………………………………………. 57

4.6.3 – Prey size …………………………………………………………………………………………………………………. 58

4.6.4 – Hydration ……………………………………………………………………………………………………………….. 58

4.6.5 – Effect of aging ………………………………………………………………………………………………………… 59

4.6.6 – Pollen consumption ………………………………………………………………………………………………… 59

4.6.7 – Effect of reproduction on web construction …………………………………………………………… 59

4.7 – Light intensity experiment (Zygiella x-notata) …………………………………………………………………… 62

4.8 – Hierarchy of variation ……………………………………………………………………………………………………….. 64

4.9 – Lack of stabilimenta ………………………………………………………………………………………………………….. 65

4.10 – Preliminary observations of stabilimenta ……………………………………………………………………….. 66

4.11 – Conclusion ……………………………………………………………………………………………………………………… 67

Appendix ……………………………………………………………………………………………………………………….. 69

References …………………………………………………………………………………………………………………….. 73

Figures, tables and images:

Chapter 1 – Introduction and background Image 1.1 & 1.2 …………………………………………………………………………………………………………………………. 16

Image 1.3 & 1.4 …………………………………………………………………………………………………………………………. 17

Chapter 2 - Methodology Figure 2.1 & 2.2 …………………………………………………………………………………………………………………………. 20

Figure 2.3 ………………………………………………………………………………………………………………………………….. 21

Image 2.1 ………………………………………………………………………………………………………………………………….. 23

Image 2.2 & 2.3 ………………………………………………………………………………………………………………………… 24

Image 2.4 …………………………………………………………………………………………………………………………………. 25

Chapter 3 - Results Table 3.1 & Figure 3.1 ………………………………………………………………………………………………………. 28

Figure 3.2 …………………………………………………………………………………………………………………………. 29

Table 3.3 & 3.3 …………………………………………………………………………………………………………………. 30

Figure 3.3 & 3.4 ………………………………………………………………………………………………………………… 31

Figure 3.5 & 3.6 ………………………………………………………………………………………………………………… 32

Figure 3.7 & 3.8 ………………………………………………………………………………………………………………….33

Table 3.4 .…..…………….………………………………………………………………………………………………………. 34

Figure 3.9 & 3.10 ……….…………………………………………………………………………………………………….. 35

Table 3.5 & 3.6 …………..…………………………………………………………………………………………………….. 36

Figure 3.11 & 3.12 …………………………………………………………………………………………………………….. 37

Figure 3.13 & 3.14 …………………………………………………………………………………………………………….. 38

Figure 3.15 & 3.16 …………………………………………………………………………………………………………….. 39

Table 3.7 ……………………………………………………………………………………………………………………………. 40

Table 3.8 ……….…………………………………………………………………………………………………………………… 41

Table 3.9 & Figure 3.17 ……………………………………………………………………………………………………… 42

Figure 3.18 & 3.19 ……………………………………………………………………………………………………………… 43

Figure 3.20 & 3.21 ……………………………………………………………………………………………………………... 44

Figure 3.22 & 3.23 ………………………………………………………………………………………………………...... 45

Figure 3.24 & 3.25 ………………………………………………………………………………………………………………. 46

Figure 3.26 ………………………………………………………………………………………………………………………….. 47

Table 3.10 …………………………………………………………………………………………………………………………… 48

Figure 3.27 …………………………………………………………………………………………………………………………. 49

Chapter 4 – Discussion Image 4.1 ……………………………………………………………………………………………………………………………. 60

Image 4.2 & 4.3 …………………………………………………………………………………………………………………… 61

Image 4.4 ……………………………………………………………………………………………………………………………. 62

Chapter 1 - Introduction and background

1.1 - Background on foraging method variation in the natural world

Much intrigue has been evoked by the way in which organisms alter foraging/prey capture techniques in response to environmental change. Changes in environmental abiotic factors such as light intensity, temperature and humidity can influence foraging effort in many species. There is limited knowledge on how animals alter their foraging technique/activity in response to varying light intensity levels. For Coccinellid beetle species, consumption rates and foraging behaviour vary in response to different visual cues, even though the same prey is hunted1. Temperature can also have an effect, as shown by South American Gastropods that show increased drilling predation on bivalve prey in response to higher temperatures existing towards the equator2. In theory, ectotherms in particular are more likely to show this response owing to increased metabolic rate at higher temperatures. Temperature can therefore increase/decrease the activity and conspicuousness of mainly ectothermic prey, which would ultimately influence predator activity. This has been shown by Eurasian Kestrel predation on Common Lizards in Norway as the probability of lizards, as opposed other endothermic prey, being delivered by kestrels to their nests tends to increase as temperatures rise towards midday when lizard activity is greater3. Recent literature has also shows that the activity of an important North American predator, the Western Rat Snake, is increased by another abiotic factor, namely humidity4. The increased activity of snakes under higher humidity leads to higher predation rates of mammals and birds. Humidity has also been shown to affect foraging activity in ants in similar fashion5. As activity levels of a predator change in response to these environmental factors, the exposure to danger, such as risk of predation to the predator itself, will fluctuate. The exposure to such dangers can cause evolutionary, physiological and/or ecological trade-offs to maximise foraging success, while being as resource efficient and undetectable as possible6.

Spiders, the focal animals in this study, are an ectothermic group of organisms where activity and predation rate may be influenced by abiotic factors. Aerial spider webs come in many forms and primarily function as a trap for mostly insect prey and a vibrational device to communicate with conspecifics7. This makes spiders ideal study organisms in regard to the impact abiotic factors have on a predator as their webs are direct indicators of their foraging effort. The orb web is the most familiar of these traps as a two-dimensional, circular meshed structure. Sticky

1 spirals are fastened onto radial threads that converge to the central hub of the web and an array of silk types are used8, 9. The evolution of the orb web is not clear and there is controversy over a monophyletic or polyphyletic origin10, 11. It is thought that Araneomorph spiders (Arthropoda: Arachnida) alter their orb webs in a variety of forms to respond to pressures in the environment, such as climate, vegetation structure, prey preference, prey abundance, predator presence and parasitic infection12. This suggests that this web type is highly advanced in its functional plasticity.

1.2 Biotic influence on orb web construction

There has been interest in how spiders change the structure of their webs in response to varying abiotic and biotic factors to maximise foraging and reproductive success, while being as silkefficient as possible13. The biotic influence on web building has been shown in Araneus diadematus, which has been observed to increase the symmetry and capture area of its web in agricultural areas when compared to spiders of the same species in a more prey abundant habitat14. Thus is provided a more efficient structure for trapping prey in agricultural land where insect abundance is much lower due to the use of pesticides. This suggests that A. diadematus is willing to invest more energy into producing more silk to increase the chance of catching insects in order to feed sufficiently. A. diadematus can even modify the fine details of its web that are associated with prey capture, as this species was also observed increasing mesh spacing when exposed to larger prey items (small Drosophila to larger mosquito prey)15. Larger mesh height would presumably facilitate the trapping of larger prey rather than smaller and a larger web can be constructed with the same/less silk investment required to build a smaller, more densely woven web16, 17. However, prey taxa must be taken into consideration when evaluating the effectiveness of larger mesh spacing on capturing larger prey items as deer flies (Chrysops sp.) are retained for less time in the orb webs of Argiope with large mesh spacing, whereas hanging flies (Hylobitacus sp.), which differ greatly in their morphology, show no difference in their retention times in webs with smaller or larger mesh18. This effect of prey availability on web construction may provide an explanation as to why Larinoides cornutus was observed in vitro building smaller, rounder orb webs with shorter spiral lengths and fewer, thinner radial threads than spiders in the field19. Reduced silk investment in a controlled environment may be linked to prey abundance and spider condition, as prey tends to be more abundant in the field

2 and impacts the web more frequently19. In the wild, spiders would therefore need to build more and thicker radii to sufficiently dissipate the kinetic energy of prey impact, also making the web more robust to damage by abiotic factors, such as wind. Reducing the frequency of prey available to orb weavers has also been shown to result in an increase in the overall web area, perhaps as an adaptation to increase the chance of catching prey when it is less abundant20.

1.3 - Spider condition

The physical condition of the spider can also be influential on web building. Spiders close to the end of their life expectancy naturally have reduced health, which causes a decline in mobility and coordination when building webs, resulting in an ‘untidy’ arrangement of spiral turns between radii21. These older spiders can also exhibit reduced silk investment, particularly in flagelliform spiral silk that is associated with prey capture, which would hinder foraging success. If orb weaving spiders are missing two or more legs, it does not appear to impact their web building and foraging success significantly. However, if they are missing three or more legs, this can result in an increased asymmetry in their orb webs22, which may negatively affect predatory success. Spider mass has previously been shown to influence web building behaviour, an example being orb weavers which tend to reduce silk investment in web building as they mature (and increase in mass) thus to preserve energy for reproductive processes23, 24. However, later studies concluded that although heavier spiders reduce their silk investment in the orb web, more energy is required to build it25. This is potentially due to the increased impact of gravity on mobility in larger spiders over a larger area, even if silk investment is reduced.

1.4 - Web building influenced by predators

One of the main influences on foraging behaviour in the natural world is predation. Foraging effort can be reduced in the presence of a predator to reduce detectability. This appears to apply to spiders and their web building behaviour. For example, Argiope versicolor is known to reduce the total area of its orb web and to increase mesh spacing in the presence of a visually acute predator26, which may reduce visibility of the web. A smaller web would enable the spider to reach captured prey with less movement, which would reduce the chance of attracting the

3 attention of a predator27. Smaller webs perhaps save energy for other behaviours, such as moving between both sides of the orb web and web shaking to blur the spider’s appearance28. However, reduced web area would result in a smaller capture area, negatively impacting prey capture rate. So it seems that, at least in some orb weaving species, there is a trade-off between foraging success and conspicuousness to predators. Also, barrier webs, three-dimensional tangled silken structures positioned in front of both sides of the orb web, are frequently built by Nephila species. These structures are thought to protect the spider from predatory attacks, with smaller, more vulnerable spiders building denser barrier webs compared to larger sexually mature adults29.

Infection by parasitoid wasps, which are frequent predators of arachnids, can alter an orb weaver’s web building for self-benefit. Spiders infected with parasitoid wasp larvae have previously been observed reducing the number of sticky spirals spun during web building30. This may benefit the parasite during pupation by reducing the chance of prey being captured and damaging the web, which suspends the wasp cocoon out of the reach of predators. Preventing the spider from investing in flagelliform spiral silk, which accounts for the majority of total web mass, may maintain spider weight to improve nourishment of the parasite31.

1.5 - Wind

The abiotic influence of the environment on web building occurs less frequently in the literature, but the few studies that are present have produced findings of interest. One abiotic factor that has been of much focus, is the effect of wind on web architecture. It has been shown that orb weavers can adjust the properties of their silk, as well at the geometry of the web in response to wind exposure. This has been shown in two Cyclosa species from Taiwan, where the seashore-dwelling C. mulmeinensis appears to build fewer radial threads, larger mesh spacing and smaller catchment area than the interior forest-dwelling C. ginnaga32. This study suggests that the building of smaller, less dense webs by C. mulmeinensis is an adaptation to reduce drag on the web in a windy coastal environment, thus preventing web damage. Larger mesh spacing is also considered to be more efficient in the catching of larger insects, which are able to fly in windier conditions, as less silk is used. The major ampullate (MA) silk in the webs

4 of C. mulmeinensis, a silk type that has been of much engineering interest due to its extraordinary tensile strength33, was shown to be significantly stronger than the MA silk of C. ginnaga. This is thought to be another adaptation to windy conditions, by perhaps investing more energy into making stronger radii than producing more radii. C. ginnaga built larger, more densely woven webs in its forest habitat where wind speed is lower and more constant, suggesting that this species may be able to invest more energy into building a more formidable trap in this habitat. Araneus diadematus behaves in a similar manner, showing reduced radii and capture area, but increased mesh spacing and reduced mesh number when exposed to windy conditions34. This study additionally tested the impact of wind on the eccentricity of webs and found that when weights were applied to wind exposed webs that were orientated horizontally, the web deformed significantly less from the horizontal plane, compared to control webs that were not exposed to windy conditions. Building a web like this would hinder prey capture capabilities, but would limit damage in a windy environment. This suggests that it may be more energy costly to rebuild a damaged web than to consume fewer prey items. Furthermore, more recent research has indicated that orb weaving spiders increase the volume of glue droplets on the capture spirals of their webs, while positioning these droplets closer together, to prevent desiccation in windy conditions35. The glue droplets constitute an important web component that is essential for prey capture and if these glue droplets dried to too great a degree, the web would lose its stickiness.

1.6 - Light

There have been previous studies focusing on the impact of light intensity on web building behaviour. Neoscona crucifera is known to significantly reduce the average radius of its web after choosing to position the web near the brightest of four artificial lights in the field. Prey was most abundant at the brightest light, suggesting that the spiders chose to build webs in the most resourceful area where they could reduce silk investment owing to the increased chance of catching prey36. Similar results occurred from a study on Larinoides cornutus, which also showed supporting evidence that adult females are genetically predetermined to select brightly lit web building sites as both wild and lab reared individuals exhibited the same behaviour in this respect37. Later studies found a similar relationship between light intensity and web building, but unlike N. crucifera, Argiope keyserlyngi showed a significant increase in overall

5 web area when exposed to higher environmental light intensity in the lab38. Silk also reflects light intensely39, rendering the orb web more visible to a wide range of predators in bright conditions40. Orb weavers spend much of their time motionless in a retreat or at the hub of the web41, however they are most vulnerable to predation when highly mobile during web building42. It is assumed that the duration of mobility when building a smaller web in brighter conditions would be shorter, reducing the chance of being detected.

Furthermore, spiders have been documented as selecting brighter, open areas over sheltered areas in which to build their webs in the field and, in a sheltered habitat, will even position their webs to face an open space43. This makes ecological sense, as flying insects tend to be more frequent in bright, open areas, such as the edge of woodland, where prey interception by the web is more likely44. However, other studies have indicated that frequently building webs in this type of environment is a genetically derived character45.

1.7 - Temperature

Although the geometric influence of temperature on webs has been poorly studied, the few studies that have been carried out have generated considerable results on this topic. Temperature has been shown to impact the geometry of orb webs, as spiders have been observed increasing web area and mesh spacing when exposed to cooler conditions from a warmer control condition34. This enables a larger web to be built, while investing less energy in producing flagelliform spiral silk. However, as spiders are ectothermic, cooler temperature may just simply hinder levels of activity, causing the spider to produce less silk.

Other studies have shown that temperature can affect web building site selection. When given the choice of multiple environmental temperatures, the sheet web-building Theridiid Archaearanea tepidoriorum selected areas to build webs where the ambient temperature was approximately 20°C46. This study subsequently found that the spiders were able to build the heaviest webs when exposed to this temperature and that web mass increased with strand density. Increased strand density is known to positively correlate with prey capture success47,

6 suggesting that spiders can select environments to build their webs where foraging is most lucrative.

Furthermore, the physical properties of silk can be affected by changes in temperature as the MA silk of Nephila edulis is known from lab experiments to significantly increase in tensile strength at very low temperatures (-60 to 0°C) and then drastically decrease at very high temperatures (~70°C)48. However, it is highly unlikely that spider silk in nature would be exposed to these extreme temperatures.

1.8 - Humidity

Although humidity has not been previously shown to clearly affect web geometry, evidently, it can impact the physical properties of flagelliform spiral silk, which has a primarily prey capture function. For example, the viscous glue droplets within flagelliform silk are known to absorb more water in humid environments, causing the glue droplets to expand, increasing surface area contact with prey and amount of air drag, which acts to dissipate prey impact emergy49, 50. This then led to studies focusing on the effectiveness of stickier webs in humid environments. In one such study, house flies placed in the webs of Araneus marmoreus constructed in 55% relative humidity (RH) were retained eleven seconds longer than in webs built in 37% RH51.

MA silk has been observed supercontracting52. This is where, in humid conditions, water causes MA thread length to shrink up to 50% and increase in volume. This silk type can supercontract at approximately 70% RH at 20°C, causing the web to increase in tension53. This tautening of the web via supercontraction has been considered an adaptive function to limit unwanted web deformation under the weight of liquid water or following prey damage54. Wet MA silk in the webs of Argiope spiders under high humidity has been shown to reduce in stiffness and extend 40% more than dry webs when prey impacts the web55. This may make sense in regard to prey capture as significantly more web deformation, in response to prey impact, could reduce deceleration, which would ultimately reduce web damage as the web would need to cope with lower peak forces. More recent research suggests that supercontraction of MA silk may have a

7 sensory function, as well as a mechanical function. Constrained supercontraction of MA silk generates a stress of approximately 50MPa on these threads, which increases the speed and amplitude of transverse waves that are important for detecting prey and conspecifics, compensating for the ‘sagging’ deformation of the web under the weight of additional water56. In theory, this would also limit the strain that major ampullate threads must withstand from kinetic energy produced by prey impacting the web57. Before web building even takes place, >70% RH is the key environmental trigger for web building in Pasilobus spiders, which is perhaps an adaptation for build in conditions when the stickiness and capture performance of the web is maximised58.

1.9 - Intraspecific web variation

When studying the impact of the environment on web building behaviour, it is important to consider the fact that web variation can occur among individuals of the same species. This is particularly evident at different ontogenetic stages. For example, juvenile Clitaetra irenae are known to build rounder, more typical orb webs, but as they mature their webs become increasingly elongated vertically to form a ‘ladder’ web59. Furthermore, ontogenetic web changes have been shown in Uloborus spiderlings, which immediately leave the egg sac after hatching to build an orb web. These early webs differ from adult webs in that they contain more radii and spiderlings only build the auxiliary spirals, which are used to guide capture spiral construction60, with a lack of genuine capture spirals61. Uloborus spins cribellate silk, which requires a fully formed cribellum and calamistrum. Spiderlings only obtain these cribellate silk spinning organs after their second moult, which may explain the lack of spirals62. Hub symmetry can also be influenced by ontogeny and this is most apparent in larger, mature spiders, which tend to orientate the hub near the top of the web, exposing a larger catchment area in the lower web portion where prey is more likely to be captured. It has been hypothesised that this is an adaptation to enable heavier spiders to move with minimum energy expenditure in a downwards direction with gravity, rather than against it63. Moreover, ontogeny can influence the structural properties of webs. Neoscona arabesca was observed investing more in the size of glue droplets on capture spirals as the spiders grew over time, which improves the performance of catching and retaining larger prey64. This could be due to a requirement of

8 much larger prey during the later stages of a spider’s ontogeny to ensure successful reproduction65.

In many orb weavers, the central nervous system (CNS) of newly hatched spiderlings only contains neuroblasts and is still not fully developed64. This may explain why the first few webs that are built by young spiders of some species are asymmetric and become increasingly symmetric over several subsequently built webs while the CNS develops. The variability in the webs of 1st instar Nephilengys cruentata is also much higher than in the webs built later in their ontogeny66. Further details of the plasticity of web architecture by juvenile spiders has been shown by Nephila madagascarienesis, at instars one and two, which construct multiple types of radii, which are defined by their division and different attachment points to the periphery of the web67. The use of these radii types was variable between individual spiders.

In some species, such as Wixia abdominalis, intraspecific web variation occurs among adults as the construction of a missing sector, with a signal thread running through this sector, is optional and can vary from spider to spider. However, this species tends to build more homogenous mesh spacing in the lower portion of missing sector webs, which is thought to improve prey retention68. This extended retention time enables the spider to reach prey before it escapes while travelling further distances across the web from a retreat at the periphery. This study further showed intraspecific variation in the presence of the sector, as some adults built complete webs with no sector where the spider occupied the hub. These webs had more heterogeneous mesh spacing, indicating that retention time is not prioritised by the spider as the distance to prey is much shorter. Flexibility in the use of a missing sector and sector orientation is also known to occur in other genera, such as Zygiella69. Furthermore, Araneus diadematus has been observed building ‘pilot webs’ when introduced to new web building sites. These initial pilot webs are usually smaller, less planar and contain fewer sticky spirals that subsequent webs, which become larger, are more planar and possess stickier spirals70. From these findings, it was suggested that A. diadematus build low investment pilot webs to assess capture success and web damage at a new site, before investing more energy in ‘proper’ subsequent webs and that the anchor points from the pilot web are used as reference points when constructing a new web. Many experiments that investigate web building behaviour

9 involve purposely destroying webs to ensure that a sample spider builds a new one. However, orb weavers of the same species that are left undisturbed to ingest their entire webs when rebuilding tend to build subsequent webs with a larger area, indicating that the amount of web remnants that can be ingested when rebuilding is energetically important for spiders and can cause intraspecific and intra-individual web geometry variation71. As with all species, there are morphological differences from one individual to another. In spiders, some individuals will naturally be larger and heavier with greater leg spans than other individuals as fully grown adults of the same species. This automatically causes variations in web area for example, as the area of a web tends to increase with spider size72.

1.10 - Other factors affecting web building

Web building experience, rather than developmental ontogeny of the CNS, can also be influential on the symmetry of orb webs. One study showed that rearing spiders in small boxes, where webs could not be built, before allowing them to build webs in larger spaces impacts their ability to build an efficient prey capture web design, as spiders reared in larger spaces, where they could build webs for a longer duration, built more asymmetric webs. The study also found that when prey was placed in the lower section of the web over a 6-day period, the experienced spiders augmented the area of the lower web portion, presumably in anticipation of prey being caught here73. In contrast, Zygiella x-notata that were reared in small boxes (where they could not build a web) for an extended period built more asymmetric webs when entered into a larger web building space. These asymmetric webs had longer lower web sections than the webs of the control spiders that were immediately entered into a web-building frame after collection74. These findings may indicate that web symmetry could have an element of species-dependant variability.

Web geometry can also fluctuate with web repair. Web repair, according to recent literature, seems to be triggered by damage of anchor threads. Breakage of these structurally supportive threads causes a great loss of capture area, which seems to trigger the quickest web repair response by Araneus diadematus75. The repair response to anchor thread damage was variable in this study, as some spiders built a new anchor thread, while others attached the broken thread to existing anchor threads to increase web tension.

10

1.11 - Stabilimenta – a prey attractant?

In addition to the orb web, some species construct stabilimenta/web decorations in the central vicinity of the web. The function of stabilimenta is thoroughly debated with several proposed theories. It was originally thought that stabilimenta serves as additional support for the web, hence the name of the structure76, 77. However, since then a multitude of theories have been proposed. These include advertisement to larger animals to prevent web destruction78, camouflage79, the provision of a ‘sunshield’ for thermoregulatory purposes80 and UV reflectance to attract insects towards the web81, 82.

The UV reflectance theory has received much attention. Argiope spiders have been shown to reduce the area of stabilimenta under high light intensity and increase stabilimenta area in low light intensities83. This may support the UV reflectance theory, as the spider can afford to invest less in stabilimenta owing to high UV reflectance of the structure and visibility to insects in bright conditions. Insect activity also tends to be higher in bright conditions as temperature usually positively correlates. This would increase the chance of prey capture and reduce the necessity of an extra prey attractant. A later field study on this topic showed supporting results for the theory. Prey capture in the webs of Argiope bruennichi with stabilimenta and webs without stabilimenta were compared, and it was found that decorated webs captured 331 prey items, while undecorated webs captured 119 (total of 450 prey items between both webs) during five hour trials per day over 10 days. This study also found that webs with stabilimenta captured more than twice the amount of large prey (>5mm) than webs without stabilimenta, which indicates that stabilimenta may be more effective in catching larger prey84. Early and recent studies have shown coherent results to support this prey luring function of stabilimenta via UV reflection85, 86, but recent studies have also found no difference in prey capture between decorated and undecorated webs87.

The morphology of web decorations differs between and within Argiope species. Specific stabilimenta shapes are considered more attractive to UV sensitive insects, such as certain bee species, which seem to approach cruciate stabilimenta much faster than linear stabilimenta88. It has also been suggested that there is a trade-off between constructing decorations to attract

11 prey and reducing detectability by visually acute predators that may be attracted to the structure89.

However, not all studies are coherent with the UV reflectance theory. Orthopterans, which are the main prey for Argiope bruennichi in their grassland habitat, do not respond to stabilimenta but flying insects, such as Hymenopterans and Dipterans, are more likely to respond due to their sensitivity to UV90, 91.

1.12 - Web damage prevention function of stabilimenta

Another theory that is often mentioned in the literature, but has little supporting evidence, is the web advertisement theory. This theory suggests that some orb weaving spiders adorn their webs with a stabilimentum to signal the web to larger vertebrates, such as birds, to prevent web damage92. Earlier studies monitored Argiope webs in the field, testing the effect of adding artificial paper decorations to undecorated webs on the overall web damage at the end of each day. This test resulted in decorated webs remaining more intact than undecorated webs, which may have reflected on the benefit of the signalling function78. However, this field study did not take into consideration the damage caused by prey capture and other environmental biotic/abiotic factors. It is possible that insects perceived the paper decorations as different from silken structures and avoided the webs more often, thus reducing web damage.

However, a later field study found that 16.4% of Argiope appensa on the island of Guam, where native bird species have been eradicated due to predation by the invasive Brown Tree Snake, adorned their webs with stabilimenta. This was then compared to A. appensa on three neighbouring islands where the native avian fauna remains intact. 41.9-56.9% of spiders on these neighbouring islands constructed stabilimenta, perhaps to advertise their webs in an environment with a higher density of bird species93. If web decorations have an antipredator function (see section 1.14), then these results may reflect the lack of predators on Guam.

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Experimentally damaging the web has recently been shown to encourage Argiope to increase the area and conspicuousness of web decorations over time94. These results have been associated with spiders learning that their environment causes a lot of web damage that is not caused by prey, but perhaps larger vertebrates, triggering this web building response to advertise the web more rigorously. However, these findings may be supportive of the stabilizing function, as spiders may have been enhancing stabilimenta in response to frequent web damage to strengthen the web in anticipation of damaging factors occurring again. Having said that, a mechanical function of the stabilimentum is unlikely as it tends to be placed loosely over web.

1.13 - Stabilimenta for predator avoidance

Antipredator functions have been applied to stabilimenta. Certain stabilimenta morphologies may serve different antipredator defences, as cruciate web decorations, which are built by certain Argiope species, are thought to make the spider seem larger to exceed the gape size of predators as the spider’s legs are often aligned with the ‘arms’ of the cross shaped structure95. Linear stabilimenta also appear to deter predators, as Blue Jays are known to, when given the choice, attack spiders in non-decorated webs more frequently that spiders in decorated webs96. However, it is difficult to interpret exactly what is it about the stabilimenta that deterred the birds. Juvenile Argiope tend to build a disc shaped stabilimentum, which has been associated with protection from predators. Juvenile Argiope versicolor move more frequently between both sides of the discoid stabilimentum in the presence of a predator, potentially using the structure as ‘shield’ for protection from attack. This was compared to the response of adult females of the same species, which do not always build decorations. Adult females seem more likely to drop from undecorated webs than from decorated webs in response to a predator, potentially supporting the antipredator function97. As web decorations are optionally constructed, there could be a cost-benefit scenario in response to the environment that determines stabilimenta construction.

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1.14 - Unexplained stabilimenta variation

In addition to these contradictory findings, there has been a focus on stabilimenta construction in a constant environment. Decoration construction in Argiope mascordi can be highly variable in a constant environment, alternating between linear, circular and cruciate shapes98. Construction of web decorations has also been associated with individual spiders that have consumed more prey biomass and are well nourished, as they can afford to invest in more silk99. These findings suggest that stabilimenta may not have an adaptive function, but that construction is an intrinsic behaviour.

1.15 - Aim of study

In this study, the effect of contrasting environmental light intensities on Zygiella x-notata web construction will be investigated. It is hypothesised that Z. x-notata will use light intensity as a proxy for anticipating levels of prey activity. Insects tend to be more active in high light intensities100, 101 and it is predicted that Z. x-notata will reduce the prey capture aspects (capture area, radii number and mesh spacing) of the web in high light intensities. This strategy would be silk-efficient, as less silk is used to build a smaller web while the probability of catching prey in a brighter environment is higher. This strategy would also reduce visibility to predators38. These prey capture aspects are expected to become enhanced, with the formation of an overall larger web, in dark conditions thus to increase the chance of catching prey when it would be less active. Fine detail changes in orbs web in response to light intensity are rarely studied in the literature and there is also a lack of focus on this topic in a controlled environment where external factors that may affect web building are limited. This study intends to solely focus on the effect of the abiotic environment on web building.

The extent of web building variation in Zygiella x-notata and Argiope bruennichi adults and juveniles under constant abiotic conditions will also be investigated. It is predicted that there will be no significant variation in any of the web characteristics in Z. x-notata and A. bruennichi, as the abiotic environment will remain the same with no changes in environmental cues that could be detected by the spiders. This is the first in depth study investigating web building variation in the fine aspects of the orb web, other than stabilimenta, under constant

14 abiotic conditions. This study aims to improve the current limited understanding of the degree of web building variation in a constant environment, providing insights into the extent of influence that abiotic factors have on web building or whether there is any influence at all. This will fill significant gaps in the current literature on this topic.

1.16 - Study species’

Argiope bruennichi is a large, strikingly marked orb weaving spider that originates from the Mediterranean region. The species’ rapid expansion into cooler northerly climates since the 20th century has led to colonisation of the United Kingdom, with the first record occurring in 1922 in Rye, East Sussex102. It is thought that the spider arrived in the UK via ballooning, where spiderlings deliberately produce a thread of silk to catch on wind currents that can then cause the animals to drift, while airborne, up to hundreds of kilometres103. There is also the possibility of introduction via artificial transport, such as cargo ships. Wasp Spiders in the UK mainly occur throughout Southern England in habitats such as meadows, grassland and woodland edge where they mainly catch Hymenopterans, Dipterans and Orthopterans via the use of their large elaborate orb webs104. Maturity occurs in mid-summer for both males (44.5mm body length) and females (11-15mm body length), but adult females are also present throughout autumn unlike the males that die during mating, due to female cannibalism, or soon after mating105.

Zygiella x-notata builds an orb web with, among British spiders, a distinctive missing sector106. A signal thread is constructed through this missing sector and leads to the spider’s retreat, where it stays in contact with one of the spider’s front legs107. The thread is used to detect vibrations of prey that are caught in the web, while the spider remains concealed108. Z. x-notata is often found in urban environments on artificial structures, such as window frames and fences where they tend to construct new webs daily. Mature males tend to live from late summer to autumn, whereas females can be seen all year round109.

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Image 1.1 – Adult female Argiope bruennichi..

Image 1.2 – The orb web of Argiope bruennichi with vertical stabilimentum.

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Image 1.3 – Adult female Zygiella x -notata .

Image 1.4 – The orb web of Zygiella x-notata with distinctive, but not always present, missing sector.

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Chapter 2 – Methodology

2.1 - Argiope bruennichi and Zygiella x-notata collection

Juvenile A. bruennichi were collected from April to June 2017 at Monks Brook Meadows in Southampton, UK (Grid reference: 50°56’49.3”N°122’16.4”W / 50.947033, -1.371220). All target individuals found while walking in suitable habitat were collected from their orb webs, which were constructed in low, dense vegetation. Collecting all individuals provided a representative samples size of juveniles and adults. Only adult females were collected between July and August. Mature males tend to not build webs at this life stage, as they are usually found near or within the webs of females. The habitat at this location is semi-improved mesotrophic grassland110.

Adult female Zygiella x-notata were collected from metal railings around the outside of the Life Sciences building in Bristol, UK. (51°27’31.4”N2°36’029”W / 51.458707, -2.600794) and back gardens in Bristol (51°30'12.2"N 2°35'56.5"W / 51.503349, -2.598969) and Southampton (50°56'17.4"N 1°22'46.2"W / 50.938167, -1.379500). Z. x-notata were collected from their webs at night as this species tends to be nocturnal and can be found in the hub of the web in darkness. Additional specimens were collected during daylight by use of an electronic toothbrush to lure spiders out from their retreats. Collection took place from February- September 2017.

2.2 - Maintenance

Juveniles were kept in small transparent Perspex® 7cm x 7cm x 3cm frames which were custom built (Figure 2.2). One side of the frames were enclosed with Perspex® covered in petroleum gel and the other side was closed off with Parafilm® covered in petroleum gel. The Parafilm® had holes pierced through it to allow ventilation within the enclosed space and exposure to the ambient environmental conditions. Adults were kept in larger 30cm x 30cm x 4cm transparent Perspex® frames divided by 35cm x 35cm PVC sheets covered in petroleum gel (Figure 2.1).

18

All spiders were kept in the laboratory for seven days, enabling them to build three acclimatisation webs before being used for data collection. Spiders that did not build three webs within this time period were released back into the wild. The petroleum gel was used to prevent spiders attaching silk threads to the dividers so that webs were not destroyed when taking frames out of the set up. This arrangement encourages the spiders to build an orb web perfectly within the frame111.

2.3 – Morphometrics

A Leica M205c microscope was used with the Leica application suite software to digitally measure the cephalothorax of each spider. Each spider was restrained in a small, flat petri dish while a photograph of each individual’s cephalothorax was taken together with a scale bar for future reference. Cephalothorax width was measured in millimetres and rounded to one decimal place (e.g. 3.84mm rounded to 3.8mm). Cephalothorax measurements were taken as not only does this body feature remain consistent in size until the spider moults, but it is this measurement that is also considered reliable when predicting web dimensions in orb weavers at all life stages112. The live mass of each spider was measured in milligrams to two decimal places. The live mass of spiders was measured because, as well as cephalothorax width, this has also been shown to influence aspects of orb web construction, such mesh spacing112, and potentially other orb web aspects. These measurements were repeated each time the spider moulted owing to significant increase in overall size, which is likely to affect web architecture.

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Divider Divider Divider Divider Divider Divider

Frame Frame Frame Frame

5cm

Figure 2.1 – A side view of the frame set up for adult spiders based on Zschokke & Herberstein (2005)113. Dividers were translucent and spiders were exposed to light coming from the bulbs on the sides of the chamber, which could penetrate the transparent sides of the Perspex® frames (labelled ‘frame’). Heavy weighted objects were placed at both ends of the set-up, holding the frames together tightly to prevent escapes. There were small gaps between dividers and frames that enable ventilation, but were not large enough for spiders to fit through.

4 cm

30cm

30cm

Figure 2.2 – Individual frame for adult spiders outside of the divider set up. Spiders built webs within the open space of the frames.

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Ventilation holes 3 cm

Parafilm screen

7 cm

Figure 2.3 – Individual frame for juvenile A. bruennichi. No dividers were necessary due to Parafilm® on one side of the frame and fixed Perspex® enclosing the other side.

Juveniles were fed one Drosophila fruit fly and adults were fed one Diptera species of 10- 15mm body length per newly constructed web. Adult webs were sprayed with water after photographs of webs were taken in order to hydrate the spiders. For juveniles, a pipette was used to place drops of water on the hub (centre) of the web, which the juveniles could drink from.

2.4 - Environmental conditions

Spiders were kept within their frame set up in two environmental chambers (Micro

ClimaSeriesTM Premium Ich Insect Chamber). In these chambers, the abiotic environment could be controlled. In this chamber, day time conditions were set at 20°C and 40% relative humidity (RH). The juveniles were exposed to a light intensity of 137.5µmol m-2 (195mm from light source). Adults were exposed to 54.8µmol m-2 from the left side of the frames (510mm from light source) and 137.47µmol m-2 from the right side of the frames (180mm from light source). Night time conditions were 16°C, 40% RH and total darkness. Spiders were exposed to a 15 hour light/ 9 hour darkness cycle to mimic UK summer time daylight hours and to not interfere

21 with the circadian clocks of these organisms. Such interference could have an effect on foraging behaviour and ultimately web building in orb weaving spiders114. The webs built by 17 individual A. bruennichi (10 juveniles and 7 adults) and 24 Zygiella x-notata were exposed to these conditions, which were kept constant in order to investigate the degree of web building under constant abiotic conditions.

2.5 - Light intensity experiment (Zygiella x-notata)

In order to investigate the effect of lowering light intensity on orb web building, 14 Zygiella x- notata individuals were exposed to two different environmental light intensities for two weeks each. The day time conditions in the control cabinet (bright condition) consisted of 157.3µmol m-² light intensity, 20°C and 40% RH. Night conditions consisted of 16°C, 40% RH and total darkness. Spiders were exposed to a 15h light/ 9h darkness cycle. The conditions in the experimental cabinet (dark condition) were the same, except that the day time light intensity was significantly lower at 29.6µmol m-². Webs built by each spider within a two-week period in each condition were measured and compared.

2.6 - Web measurements

All webs built by spiders in both environmental conditions were photographed against a black paper background. Bright lights were aimed at the webs from behind in order to illuminate the silk for easier photography and clearer images. The webs were photographed adjacent to a ruler within the plane of the web in order to set a scale on the software ImageJ, where the web aspects of interest could be measured accurately. To ensure that A. bruennichi individuals were rebuilding their webs each time they were sampled, the webs were dusted with crushed pollen granules after the photographs were taken. If the yellow pollen dust had disappeared the next day and a web was present, it was assumed that this was a new web as spiders take down their webs before building a new one. The new web could also be compared to the image of the previous web to ensure that it was definitely different and newly built. This dusting method was used as a means of avoiding web destruction, as previous research suggests that web

22 damage affects stabilimenta morphology115. The webs of Z. x-notata were destroyed after photographs were taken and the spiders were fed and watered. Only the anchor, bridge and frame threads were left intact to promote subsequent web building (Image 2.1).

Image 2.1 – Diagram to show location of bridge (yellow arrows), frame (red arrow) and anchor (blue arrow) threads outer edges of the orb web. These were left intact, while all other threads were destroyed after each web was sampled.

The web aspects of interest were radii number, centre area (mm2) (hub area and free zone area combined), catchment area (mm2) (area covered by flagelliform spirals), average upper mesh spacing (mm), lower mesh spacing (mm), right mesh spacing (mm), left mesh spacing (mm), upper and lower linear stabilimentum area (mm2) and stabilimentum length (mm) (Image 2.3). Mesh spacing was measured by dividing the length of the radial thread between in the inner- most spiral to the outer-most spiral (upper, lower, right or left portions of the web) by the number of flagelliform spirals that pass through this length measurement116. The radial thread measured for mesh spacing in each web portion was chosen as the most vertical radial thread in the upper and lower portions of the web (upper and lower mesh spacing) and the most horizontal radial thread in the right and left portions of the web (right and left mesh spacing). For discoid stabilimenta that were built by some juveniles, only area was measured due to the irregular shape of the structure and difficulty in identifying an accurate diameter. Linear

23 stabilimenta lengths were measured from the upper-most points of the upper and lower stabilimenta to the lower most point.

Upper stabilimentum

Lower Discoid stabilimentum stabilimentum

Image 2.2 – Photographs of juvenile discoid stabilimentum (left) and adult vertical linear stabilimentum (right). Location of the lower and upper portions of linear stabilimenta in the webs of Argiope bruennichi labelled in right image. Blue bars represent a scale of 1cm.

Mesh spacing

Hub

Free zone

Radial thread Capture spiral

Image 2.3 – Diagram to show web aspects of interest. Capture area is the area covered by sticky spirals. The measurement of length between the inner-most spiral and outer-most spiral along a radial thread is divided by the number of spirals that pass through this length in order to obtain the average mesh spacing. A ruler is placed adjacent to the web in order to set a scale on Image J before digitally measuring webs. Blue scale bar represents 2cm.

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The area of the characteristic missing sector in the webs of Z. x-notata was measured to investigate the plasticity of this structure under the environmental conditions provided. The area of the space between the signal thread both adjacent radial threads (left and right of signal thread) within the missing sector was also measured to explore plasticity in the symmetry of this structure. These area measurements were taken within the two outer most spirals adjacent to the signal thread, as shown in Image 2.4. If the signal thread was constructed outside of the vertical plain, it was only possible to obtain the area of the whole sector and not the area either side of the signal thread.

Signal thread

Image 2.4 – Orb web of Zygiella x-notata with the missing sector outlined in yellow. The red line shows the limit of area measurement in the missing sector, as area measurements were taken within both of the outer most spirals adjacent to the signal thread. The area of the space between the signal thread of both adjacent radial threads (green) was also measured, again, within the red line. Blue bar represents 2cm.

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2.7 - Statistical methods

With the use of SPSS statistical software, a Friedman’s test was used to investigate the extent of variation in the web building of juvenile A. bruennichi, adult A. bruennichi and Z. x-notata over time under constant abiotic conditions. The assumptions of the Friedman’s test are that one group is measured on three or more occasions (the orb webs of spiders over 3 or more days), the group is a random sample from the population, the dependant variable (web aspects) is measured at the continuous or ordinal level and data are not normally distributed. The Friedman’s test is often considered the non-parametric version of the one-way ANOVA. The significance level chosen was 0.05.

A Wilcoxon signed-rank test was used to compare web construction by Z. x-notata in both bright and dark conditions in order to compare differences in the construction of web aspects in both conditions. For this test, the dependant variable is also measured at the continuous or ordinal level (web aspects), there must be at least one independent variable (light intensity) arranged into two or more categories (light and dark condition) to allow comparison and again, this test is non-parametric as data are not normally distributed. This test takes into account that the samples are paired and compares the difference in web aspect-building within each spider in the light and dark condition. The significance level chosen was 0.05.

Chapter 3 – Results

In total, 261 webs were built by all 41 individual spiders from both Argiope bruennichi and Zygiella x-notata in all experiments overall (Appendix – Table A1). 138 webs were built by both species in the experiments that investigated web building variation under a constant abiotic environment. 32 webs of these webs were built by all adult A.bruennichi, with 18 webs between 6 individuals used for analysis; juvenile A. bruennichi built 56 webs overall, with 40 webs between 10 individuals used for analysis and Z. x-notata built 50 webs overall, with 33 webs built by 11 individuals used for analysis. In the light intensity experiment, 123 webs were built by 14 individual Z. x-notata overall with 58 webs built in light condition (3-6 webs built by each individual) and 65 webs built in dark condition (3-8 webs built by each individual). These

26 subsections of webs used by each sample group were based on the number of webs built by spiders after the three acclimatisation webs. Post-acclimatisation webs used were then rounded down to the number of webs most frequently constructed among all spiders in each group for data analysis. This ensured that the same number of post-acclimatisation webs were analysed for each spider.

3.1 - Web building variation under constant abiotic conditions Web building variation in a constant abiotic environment was investigated to test the null hypothesis that all of the web aspects measured would not significantly vary over time under constant conditions. The Friedman’s tests show that the null hypothesis can be accepted for Zygiella x-notata as none of the web aspects measured significantly varied. However, it can be rejected for adult and juvenile Argiope bruennichi for some specific web aspects, as there was significant variation in the construction of upper mesh spacing in the juvenile A. bruennichi webs and radii number in adult A.bruennichi webs over time.

3.2 - Juvenile Argiope bruennichi

Starting with juvenile A. bruennichi, only upper mesh spacing showed marginally significant variation (N=10, χ²=8.040, p=0.045) (Figure 3.1) under constant environmental abiotic conditions (20°C daytime, 40% RH day and night, 137.5 micro moles per squared, 16°C night time, 15 hours light/ 9 hours dark). Juvenile A. bruennichi webs aspects that did not significantly vary over time include radii number (N=10. χ²=4.355, p=0.226); lower mesh spacing (N=10, χ²=4.394, p=0.222), left mesh spacing (N=10, χ²=6.184, p=0.103), right mesh spacing (N=10, χ²=5.880, p=0.118), capture area (N=10, χ²=1.800, p=0.615) and centre area (N=10, χ²=5.160, p=0.16) (Table 3.1).

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Table 3.1 – Variability of the web parameters of 10 juvenile A. bruennichi over time. Asterisk denotes significant variation over time, as determined by the Friedman’s test (p<0.05).

Web parameter Chi-square Days/webs No. of individuals P value Radii no. 4.355 4 10 0.226 Lower mesh 4.394 4 10 0.222 spacing Upper mesh 8.040 4 10 0.045* spacing Left mesh spacing 6.184 4 10 0.103 Right mesh spacing 5.880 4 10 0.118 Capture area 1.800 4 10 0.615 Centre area 5.160 4 10 0.16

Figure 3.1 – Scatter-line plot to show significant variation in upper mesh spacing within and between 10 individual juvenile Argiope bruennichi over a 4-day period under constant abiotic conditions. Each colour represents an individual spider.

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2.5

1 ) 7 2 5 4 1.5 6 25 1 23

15 Upper mesh spacing (mm spacingmesh Upper 0.98 1.13 1.08 1.16 1.05 1.76 1.22 0.5 1.36 1.09 1.05 (Av) (Av) (Av) (Av) (Av) (Av) (Av) (Av) (Av) (Av) 3 2

0

Figure 3.2 – Bar graph to show mean upper mesh spacing measurements (y-axis) for each juvenile A. bruennichi individual over a four-day period. Standard deviations, as represented by error bars, show variation in this web parameter within individuals. Each colour represents an individual spider.

Although the Friedman’s test results show that centre area, radii number and capture area did not significantly vary in the webs of juvenile A. bruennichi over a 4-day period (Table 3.1), variation in these web characteristic measurements within each individual spider (6% - 18.5% coefficient of variance range; Table 3.2; Fig. 3.3, 3.5 and 3.7) is lower than variation of these web characteristic measurements between individuals (24.03% - 28.1% CV range) on each day (although narrower CV range 24.03% - 28.1%; Table 3.3; Fig. 3.4, 3.6 and 3.8) when comparing coefficient of variance values (Table 3.2 and 3.3).

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Table 3.2 - Coefficient of variance, expressed as a percentage, within each individual juvenile A. bruennichi for centre area, radii number and capture area measurements over the 4-day period (n=4 for each cell).

Spider 1 7 5 4 6 25 23 15 3 2 Average Centre 15.4% 22.4% 6.3% 11.3% 5% 22% 11.8% 10.1% 22.7% 22.2% 14.92% area

Radii 10.3% 13.2% 8.4% 6.6% 7.6% 7.3% 8.9% 4.4% 12% 11.3% 9% no.

Capture 19.7% 17.8% 7.9% 17.4% 9.3% 18.5% 9.9% 3.5% 16.9% 22% 14.29% area

Average 15.13% 17.8% 7.53% 11.77% 7.3% 15.93% 10.2% 6% 17.2% 18.5%

Table 3.3 - Coefficient of variance percentages among all individual juvenile A. bruennichi centre area, radii number and capture area measurements on each of the 4 days (n=10 for each cell).

Day 1 2 3 4 Av Centre area 49.7% 43% 40.8% 51.3% 46.2 Radii no. 10.4% 18.8% 16% 18.1% 15.83 Capture area 22.7% 21.7% 16.1% 14.9% 18.85 Av 27.6 27.83 24.03 28.1

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Figure 3.3 – Whisker plot to show considerable centre area variation within each individual juvenile A. bruennichi over a 4-day period (n=4). Each colour represents an individual spider.

Figure 3.4 – Whisker plot to show centre area variation among all individual juvenile A. bruennichi on each of the 4 days (n=10).

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Figure 3.5 – Whisker plot to show considerable radii number variation within each individual juvenile A. bruennichi over a 4-day period. Each colour represents an individual spider.

Figure 3.6 – Whisker plot to show radii number variation among all individual juvenile A. bruennichi on each of the 4 days appears to be less considerable than variation within individuals.

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Figure 3.7 – Whisker plot to show considerable capture area variation within each individual juvenile A. bruennichi over a 4-day period. Each colour represents an individual spider.

Figure 3.8 – Whisker plot to show capture area variation among all individual juvenile A. bruennichi on each of the 4 days appears to be less considerable than variation within individuals.

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3.3 - Adult Argiope bruennichi Adult female A. bruennichi showed slightly different variations in web building over time, as radii number significantly varied over time (N=6, χ²=8.667, p=0.013) (Figure 9) and upper mesh spacing did not (N=6, χ²=0.273, p=0.873). However, like juvenile A. bruennichi, adult female A. bruennichi did not show any significant changes over time in lower mesh spacing (N=6, χ²=0.667, p=0.717); left mesh spacing (N=6, χ²=1, p=0.607), right mesh spacing (N=6, χ²=0.261, p=0.878), capture area (N=6, χ²=4, p=0.135) and centre area (N=6, χ²=4.333, p=0.115) (Table 3.4).

Table 3.4 – Variability of the web parameters of 6 adult A. bruennichi over time. Asterisk denotes significant variation over time, as determined by the Friedman’s test (p<0.05).

Web Species Chi-square Days/webs No, of P value parameter individuals

Radii no. Argiope bruennichi 8.667 3 6 0.013* Lower mesh Argiope bruennichi 0.667 3 6 0.717 spacing Upper mesh Argiope bruennichi 0.273 3 6 0.873 spacing Left mesh Argiope bruennichi 1.000 3 6 0.607 spacing Right mesh Argiope bruennichi 0.261 3 6 0.878 spacing Capture area Argiope bruennichi 4.000 3 6 0.135 Centre area Argiope bruennichi 4.333 3 6 0.115

34

36

34

32 22 30 17

28 30

26 32 19 24 16 22

20 0 0.5 1 1.5 2 2.5 3 3.5 4

Day/Web

Figure 3.9 – Scatter plot to show significant adult female A. bruennichi radii number variation within and between 6 individuals over a 3-day period under constant abiotic conditions. Each colour represents an individual spider. Plots for spider 22 and 30 are identical.

40

35

30 22 17 25 30 20 27.3 31.7 27.3 26.3 3.6 29.67 32 Av Av Av Av Av Av 15 19

10 16

5

0

Figure 3.10 – Bar graph to show averages (y-axis) of adult A.bruennichi radii number counts over a 3-day period. Standard deviations, as represented by error bars, show variation in number of radial thread built by 6 individuals over 3 days. Each colour represents an individual spider.

Although the Friedman’s test results show no significant variation in upper mesh spacing, centre area and capture area between adult A. bruennichi over a 3-day period (Table 3.4),

35 variation in these web characteristics appear to be low overall within the web building of each individual spider. However, some spiders show much higher CV values than others according to the coefficient of variation values for these web aspects (CV range: 9.77% - 53.87; Figures 3.11, 3.13 and 3.15). Variation in these web parameters among all individuals over the 3-day period appears to be higher overall with a narrower CV range (CV range: 34.07% - 44.43%; figures 3.12, 3.14 and 3.16). Upper mesh spacing and centre area variation is at its highest on day 2 (Figures 3.12 and 3.14).

Table 3.5 - Coefficient of variance percentages within individual adult A. bruennichi for upper mesh spacing, centre area and capture area measurements over the 3-day period.

Spider 22 17 30 32 19 16 Average Upper 10.2% 24.1% 14% 19.5% 6.6% 22.2% 16.1% mesh

Centre 26.8% 20.9% 10.3% 13.9% 19.8% 16.6% 18.1% area

Capture 18.1% 3.8% 5% 36.7% 8.3% 45.2% 19.5% area

Average 18.4% 16.3% 9.8% 23.4% 34.7% 53.9

Table 3.6 - Coefficient of variance percentages between all individual A. bruennichi upper mesh spacing, centre area and capture area measurements on each of the 3 days.

Day 1 2 3 Average Upper mesh 13.8% 29.8% 29.5% 24.4% Centre area 53.8% 29.8% 62.7% 48.8% Capture area 50.4% 42.6% 41.1% 44.7% Average 39.3% 34.1% 44.4%

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Figure 3.11 – Upper mesh spacing variation within each individual adult A. bruennichi over a 3-day period. Each colour represents an individual spider.

Figure 3.12 – Upper mesh spacing variation among all adult A. bruennichi over the 3-day period appears to be less considerable than variation within individuals, with most variation occurring on day 2.

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Figure 3.13 – Centre area variation within each individual adult A. bruennichi over a 3-day period. Each colour represents an individual spider.

Figure 3.14 – Centre area variation among adult A. bruennichi over the 3-day period appears to be less considerable than variation within individuals, with most variation occurring on day 2.

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Figure 3.15 - Capture area variation within each individual adult A. bruennichi over a 3-day period. Each colour represents an individual spider.

Figure 3.16 – Capture area variation among all adult A. bruennichi over the 3-day period appears to be less considerable than variation within individuals.

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3.4 - Stabilimenta building

Stabilimenta building in juvenile and adult A. bruennichi was scarce, occurring only 16 times between juveniles and adults within 8 individuals (4 times in adults between 2 individual adult A. bruennichi and 12 times between 6 juveniles).

3.5 - Zygiella x-notata

Zygiella x-notata adult females showed no significant variation in any of the web parameters measured over time: Radii number (N=11, χ²=3.659, p=0.161); lower mesh spacing (N=11, χ²=3.455, p=0.178), upper mesh spacing (N=11, χ²=2.182, p=0.336), left mesh spacing (N=11, χ²=3.818, p=0.148), right mesh spacing (N=11, χ²=5.091, p=0.078), capture area (N=11, χ²=0.727, p=0.695), centre area (N=11, χ²=3.455, p=0.178), sector area (N=11, χ²=1.333, p=0.513) (Table 3.7).

Table 3.7 – Friedman’s test results show no significant web building variation over time for adult female Zygiella x-notata web parameters (p>0.05).

Web Species Chi-square Days/webs No. of P value parameter individuals Radii no. Zygiella x-notata 3.659 3 11 0.161 Lower mesh Zygiella x-notata 3.455 3 11 0.178 spacing Upper mesh Zygiella x-notata 2.182 3 11 0.336 spacing Left mesh Zygiella x-notata 3.818 3 11 0.148 spacing Right mesh Zygiella x-notata 5.091 3 11 0.078 spacing Capture area Zygiella x-notata 0.727 3 11 0.695 Centre area Zygiella x-notata 3.455 3 11 0.178 Sector area Zygiella x-notata 1.333 3 11 0.513

Although the Friedman’s test results show no significant variation in any of the Zygiella xnotata web characteristics measured over time, there is low overall variation in majority of web building characteristics within individual spiders over a 3-day period, with some individuals

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showing much higher variation (Table 3.8, CV range: 7.09% - 45.5%). These web characteristics include centre area (CV range for centre area: 4.4% - 39.6%; Figure 3.17) and capture area (CV range for capture area: 5.2% - 50%; Figure 3.19), which are also low in overall variation within individual adult and juvenile A. bruennichi. Radii number also has low variability within individual Z. x-notata, (CV range for radii number: 2.7% – 20%, Figure 3.21) and upper mesh spacing has low overall variability with much larger CV values shown in some spiders (CV range for upper mesh: 7.6% - 43.8%, Figure 3.23), which is the same for adult A. bruennichi (Figure 3.11) within individuals. Z. x-notata showed low overall variation within individual left, right and lower mesh spacing over the 3 days with some individuals showing much high variation than others (appendix: Figure A1, A3, A5). The unique Z. x-notata web feature, the missing sector, showed very high area variation within some individual spiders, while other individuals sustain more consistent sector areas (CV range for sector area: 10.5% - 173.2%, figure 3.25). The variation in all of these web characteristic measurements between individuals on each of the 3 days is high (CV range: 32.63% - 52.96%; Figures 3.18, 3.20, 3.22, 3.24, 3.26 and appendix: Figure A2, A4, A6 respectively). These observations are particularly apparent when comparing coefficient of variance values (Table 3.8 and 3.9).

Table 3.8 – Coefficient of variance percentage values for radii, left mesh, right mesh, lower mesh, upper mesh, capture area, centre area and sector area measurements within individual Z. x-notata over the 3-day period.

Spider 40 34 30 32 17 13 24 31 33 42 66 Average

Radii no. 7.1 13. 6 13. 2 10.2 4.5 5.8 8.3 20 4.2 2.7 4.7 8.57

Left mesh 9.1 32. 3 28. 8 28.6 44.6 9.8 20.2 30.2 30.2 4.3 10.1 22.65

Right 3.6 8.1 21. 3 47.9 26.2 8.8 11 30.9 11.5 15.7 9.9 17.72 mesh Lower 7.3 19 41. 7 29.4 10.2 15.4 11.7 33.75 4.5 24.6 10.4 18.90 mesh Upper 7.6 25. 6 43. 8 35.2 41.2 17.3 18.6 26.9 10.6 11.6 24 23.85 mesh Capture 7.1 8.2 50 27.9 26.6 21.5 5.2 46.7 40.3 33.8 8.9 25.11 area Centre 4.4 25. 9 49. 4 11.9 20.4 7.2 20.2 20.4 26.8 39.6 36.5 23.88 area Sector 10.5 33. 2 44. 6 173.2 57.7 31.7 86.6 19.3 33.1 173.2 118.1 71.02 area Average 7.09 20. 74 36. 45.54 28.9 14.6 22.7 28.52 20.1 38.18 27.83 60 3 9 3 5

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Table 3.9 – Coefficient of variance percentage values for web characteristic measurement between individual Z. x- notata on each of the 3 days. Day 1 2 3 Average Radii no. 21.3 18 19.4 19.57 Left mesh 22.9 37.2 17.7 25.93 Right mesh 24.3 56.2 33.6 38.03 Lower mesh 17.2 40 32.3 29.83 Upper mesh 39 37.2 28.8 35 Capture area 33.1 71.2 54.3 52.87 Centre area 31.8 47 55.1 44.63 Sector area 71.4 116.9 58.8 82.37 Average 32.63 52.96 37.5

Figure 3.17 – Considerable centre area variation within each individual adult Z. x-notata over a 3-day period. Each colour represents an individual spider.

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Figure 3.18 – Centre area variation among all adult Z. x-notata on each of the 3 days is less considerable than variation within individuals.

Figure 3.19 – Considerable capture area variation within each individual adult Z. x-notata over a 3-day period. Each colour represents an individual spider.

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Figure 3.20 - Capture area variation among all adult Z. x-notata on each of the 3 days is less considerable than variation within individuals.

Figure 3.21 - Considerable radii number variation within each individual adult Z. x-notata over a 3-day period. Each colour represents an individual spider.

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Figure 3.22 – Radii number variation among all adult Z. x-notata on each of the 3 days is less considerable than variation within individuals.

Figure 3.23 - Considerable upper mesh spacing variation within each individual adult Z. x-notata over a 3-day period. Each colour represents an individual spider.

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Figure 3.24 – Upper mesh spacing variation among all adult Z. x-notata on each of the 3 days is less considerable than variation within individuals.

Figure 3.25 - Considerable sector area variation within each individual adult Z. x-notata over a 3-day period. Each colour represents an individual spider.

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Figure 3.26 – Sector area variation among all adult Z. x-notata on each of the 3 days is less considerable than variation within individuals.

3.6 - Light intensity experiment A Wilcoxon signed-rank test was used to investigate differences in web building in response to different environmental light intensities. The results show that there was no significant difference between the web parameter measurements in the webs of 14 individual Z. x-notata in the light condition versus the dark condition, except for lower mesh spacing (Table 3.10). This rejects the proposed hypothesis that capture area, radii number and all mesh spacing in the webs of Z. x-notata would be reduced in the high light intensity condition and enhanced in the low light intensity condition and the null hypothesis can be accepted as light intensity had no effect on web construction apart from lower mesh spacing. However, 9 out of 14 spiders, although marginally, significantly increased the lower mesh spacing in the brighter condition (Table 3.10; Figure 3.27).

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Table 3.10 – Differences in web parameter measurement from 14 individual Zygiella x-notata adult female webs in both the light and dark condition. Results of the Wilcoxon Signed Rank test show no significant differences in between the web parameters built in the light and dark condition, except for lower mesh spacing (p=>0.05 for all web parameters except lower mesh, which shows p=0.048*). Overall mesh spacing is the mean of upper, lower, right and left mesh spacing measurements for each spider in the light and dark condition.

Web Species Test N P value Mean (L) Mean (D) SD (L) SD (D) parameter statistic (Z) Radii no. Zygiella -1.224b 14 0.221 26.7 28.47 5.58 3.77 x-notata Lower mesh Zygiella -1.977b 14 0.048* 2.71 2.43 0.62 0.37 spacing (mm) x-notata Upper mesh Zygiella -8.47b 14 0.397 3 2.96 0.7 0.8 spacing (mm) x-notata Left mesh Zygiella -1.287b 14 0.198 2.64 2.42 0.71 0.46 spacing (mm) x-notata Right mesh Zygiella -0.910b 14 0.363 2.61 2.48 0.51 0.369 spacing (mm) x-notata Overall mesh Zygiella -1.601b 14 0.109 2.74 2.57 0.57 0.43 spacing (mm) x-notata Capture area Zygiella -0.659b 14 0.510 10577 11199.58 4439.61 3294.99 (mm2) x-notata Centre area Zygiella -.910b 14 0.363 895.95 984.19 324.78 312.68 (mm2) x-notata Sector area Zygiella -1.475b 14 0.140 694.35 590 333.92 257.26 (mm2) x-notata

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The affect of light intensity on lower mesh construction by Zygiella x-notata 4.5

L 69 4 71 L L 3.5 72 D L 73 3 L D D D L L L 74 L D D D D L 2.5 D L D 77 D D L L D 79 2 L D 83 1.5 89 90

Lower mesh height (mm) height mesh Lower 1 92

0.5 84 85 0

Figure 3.27 – The average values of lower mesh spacing measurements in the light intensity experiment. 9 out of 14 Z. x-notata built larger lower mesh spacing in bright light intensity, although to no significant extent. ‘L’ represents the lighter conditions and ‘D’ represents the darker condition. Each colour represents an individual spider.

Chapter 4 - Discussion

Web building in juvenile A. bruennichi, adult A. bruennichi and Zygiella x-notata was mostly consistent over time under constant abiotic conditions and remained mostly consistent in Zygiella x-notata, apart from lower mesh spacing, when exposed to different environmental light intensities. The only web parameters that significantly varied over time under constant conditions were radii number in adult A. bruennichi webs and upper mesh spacing in juvenile A. bruennichi webs. This variation in A. bruennichi upper mesh spacing and radii number rejects the null hypothesis that web building by both species would remain totally consistent under the same abiotic conditions over time. The mostly insignificant changes in web building by Z. x-notata in low and high light intensity environments somewhat accepts the other null hypothesis, thus spiders do not invest less in the prey capture aspects (smaller capture area, fewer radial threads and increased overall mesh spacing) in bright conditions when prey is

49 likely to be more active. However, the decrease in mesh spacing shown by spiders in the dark condition was marginally significant.

In order to balance foraging success and acclimatisation to laboratory conditions between spiders, three webs were allowed to be built by each spider in the frame set up before experiments took place. Allowing spiders to build three or more acclimatisation webs is considered the best practice in preparation for laboratory-based experiments in previous studies34.

4.1 - Juvenile upper mesh variation and overall mesh consistency

Orb web asymmetry has been observed in various spider species, which has led to the proposal of multiple adaptive hypotheses. The sticky capture spirals of the orb web have been shown to be more abundant and to cover a larger area below the hub in the lower portion of vertically- built orb webs in most cases117, 118. Uncommon exceptions are represented by spiders in the Scloderus genus as they tend to drastically enhance the area of their orb webs above the hub in the upper portion of the web, where more spirals reside119. This type of asymmetry shown by Scloderus is hypothesised to be an adaptation for catching moths, as a moth will often tumble down the web and lose protective wing scales when its flight is intercepted. Once enough wing scales are lost, the silk can adhere to the moth’s body and ensnare it120. Aside from area, some genera, such as Micrathena and Gasteracantha, build non-vertical orb webs with a roughly equal number of spirals in the lower and upper portions of the web121, suggesting that spiral distribution may be genera-specific. In terms of mesh spacing, earlier studies describe the mesh spacing of orb webs to be narrower in the lower portion of the web122, while later observations indicate that there is no difference between mesh spacing in the upper and lower areas of the web123. Some spiders, mostly those in the families Tetragnathidae and Uloboridae, built non- vertical webs124. Mesh spacing in the non-vertical orb webs of Micrathena gracilis tend to be smaller in the lower web region, while mesh is narrower in the upper region of Leucage mariana, suggesting that mesh spacing could be interspecifically determined among different species125. Mesh spacing has also been shown to be more uniform in the lower section of the web126. Furthermore, arrangement of radial threads is often asymmetric. The angle between

50 individual radial threads tends to be smaller in the lower and often larger part of the web as this would reduce the likelihood of sticky spirals contacting each other and fusing when displaced by wind, which prevents web damage125. Orb weavers have also been known to ‘double’ radial threads, by splitting one radial thread into two, in the upper portion of the web to structurally cope with larger forces in this area127, 128.

Although not all aspects of asymmetry were measured in this study, the significant variation in upper mesh spacing over time in the webs of juvenile A. bruennichi may relate to the ‘top/bottom’ asymmetry hypothesis, which proposes that the habit of orb weavers building a larger capture area in the lower region of the web promotes the capture of prey there. This would enable the spider to move downwards with gravity to catch prey, which is less energetically costly than moving upwards against gravity129. Juvenile A. bruennichi may not prioritise consistent upper mesh building as it is not a priority for prey capture129. Further investigation, with both asymmetry and mesh spacing being measured simultaneously over time, with similar methodology to this study, is required to understand the relationship between upper mesh spacing and the top/bottom asymmetry in the webs of orb weavers and how this relationship changes as a spider moults and grows.

The interspecific differences in the construction of mesh spacing shown in previous studies125 may support the results in this study, as no significant variation in upper mesh spacing over time occurred in the webs of Zygiella x-notata, but did occur in the webs of juvenile A. bruennichi. Perhaps these results suggest that Z. x-notata is a more consistent web-builder than Argiope bruennichi under constant conditions. The uniformity of lower mesh building observed in previous studies125 is also coherent with the results as adult A. bruennichi, juvenile A. bruennichi and Zygiella x-notata all showed insignificant variation in lower mesh construction over time. Future research should focus on differences in mesh spacing consistency between various species in order to better understand interspecific patterns in the construction of this web aspect and the possible functions that differing mesh sizes serve for different orb weavers in contrasting habitats.

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4.2 - Spatial constraints

In contrast to juvenile A. bruennichi, adult A. bruennichi did not show significant variation in upper mesh spacing over time. This may be due to the restricted size of the smaller frames that juveniles were kept in, as Araneids, including species in the Argiope genus, have been shown to alter the geometry of their webs drastically to conform to the space available within different sized frames130. Other studies have shown that Araneus diadematus elongates its web vertically when exposed to narrower web-building spaces131. This treatment also caused A. diadematus to reduce mesh size on the shorter horizontal radial threads near the left and right areas of the hub, but mesh was larger along longer, vertical radial threads above and below the hub, indicating that radii position and length influences the attachment points of the sticky spirals. Furthermore, another Araneid, Leucage argyra, is capable of constructing extremely reduced webs when kept in small frames that are only 7% of the mean span of webs in the wild for this species132. L. argyra also demonstrated changes in orb web design at different stages of web building, including radial thread and frame connection, building of the hub, layout of the sticky spirals and termination of sticky spirals. Other orb weavers, such as the ladder web spider Telaprocera maudae, significantly elongated their webs when kept in a narrow, horizontal frame space and built more circular webs when kept in frames with an increased diameter in web-building space133. This species positions its web against tree trunks in the wild and this web-building plasticity would enable any tree of any trunk size to be inhabited throughout the spider’s development and dispersal from tree to tree59. This behavioural flexibility may also aid in maximising prey capture with the available space64.

The extent of building plasticity may be species-dependant, as Cyclosa caroli showed very low web-building frequency when kept in smaller, elongated frames, whereas Eustala ilicita showed higher web-building frequency and was quick to adapt its webs to the limited space, indicating that behavioural plasticity between these two phylogenetically close species is influenced by the microstructure of the web-building area134. E. ilicita may require building flexibility, as it inhabits specific vegetation where it occurs in high densities135, whereas C. caroli occurs in low density in various understory vegetation, where it would naturally have more space and would not be accustomed to building in restricted spaces136. In summary, perhaps juvenile A. bruennichi would have kept upper mesh spacing more consistent if

52 provided with more space. It is interesting to note that lower mesh was kept consistent, which may indicate prioritisation as catching prey below the hub is more energy efficient129.

The consistent building environment of the frames may also explain the lack of variation in the majority of web parameters, apart from adult A. bruennichi radii number and juvenile A. bruennichi upper mesh spacing, over time among all sample spiders from both species under constant abiotic conditions as orb weavers can use anchor points from previously built webs at the same site as a reference for building new webs, reducing cognitive demand137. The frame size and shape remained constant throughout experiments and although webs of Z. x-notata were destroyed after they were photographed, the frame, bridge and anchor threads remained intact to promote web repair. This would have enabled the spiders to build webs in exactly the same building space where they were likely to use the frame, bridge and anchor threads from their previous webs as reference points. This may explain the overall lack of web variation in both species used in this study in laboratory conditions, but these findings may not reflect web variation in the wild, as spiders often disperse or are displaced by environmental factors in nature, increasing cognitive demand to build webs at new sites133. Furthermore, this could be why there were almost no significant differences in web building shown by Zygiella x-notata in the light intensity experiment when exposed to a high light intensity environment and a low light intensity environment as perhaps the spatial aspects of the environment, which were constant, are more influential than the abiotic conditions on web building behaviour.

Although the effect of varying web-building space on orb web geometry has been well documented, the diversity of sample species in previous studies is relatively small as there are approximately 4,500 orb weaving spider species known worldwide138. This provides vast opportunity in future research to investigate how the many different orb weaving species, which have not been used in previous experiments, adapt their web building to spatial constraints and how the extent of building plasticity over time changes between phylogenetically distant and close species that differ in their ecology.

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4.3 - Web building experience

Juvenile orb weavers are known to build circular, more symmetrical webs139, which challenges the previously mentioned ‘top/bottom asymmetry’ hypothesis. Larinoides sclopetarius adults that are deprived of web building experience have subsequently been observed building more circular, symmetrical webs much like those of many juvenile orb weavers73. Some A. bruennichi adults and juveniles in this study built webs readily and more frequently than other individuals in the frame set up. There was variation in the number of webs built between individual spiders, as although four webs from each juvenile and three webs from each adult were used for analysis, some built up to seven webs (Appendix – Table 1). Perhaps the infrequent web-builders lacked the experience required to maintain consistent web geometry and this may have influenced increased radii variation in adult webs and upper mesh variation in juvenile webs. Adult A. bruennichi that built infrequently may have shown this previously observed tendency to build more symmetrical webs when deprived of web building experience, while frequent web builders built asymmetrical webs. However, this does not explain the upper mesh variation in juvenile A. bruennichi and further investigation into mesh variation in the upper and lower regions of the orb web in experienced and inexperienced spiders is required to understand this phenomenon more thoroughly.

4.4 - Size limitation hypothesis

Another hypothesis, the ‘size limitation hypothesis’, may provide an alternative explanation for the lack of web building variation in most of the web aspects measured over time under consistent abiotic conditions in juvenile A. bruennichi, as it suggests that young animals with underdeveloped brains are limited in their behavioural flexibility140. This hypothesis also suggests that young animals, including juvenile spiders, may be more prone to be making ‘errors’ when building multiple webs over time, limiting the consistency of web geometry. This seems logical, as smaller neurones in juvenile spiders would presumably limit the complexity of behaviours141. However, it has been difficult to show convincing experimental support for this142. These previous findings may be reflected in the results from juvenile A. bruennichi building, as keeping upper mesh spacing consistent, along with the rest of the web, may have been beyond their cognitive ability while their central nervous systems are still developing. However, future experiments with similar methodology, a larger sample size of juvenile spiders 54 and a wider range of species would investigate mesh ‘errors’ in more detail and could potentially show how these errors differ interspecifically, as different species may show inconsistencies in different web characteristics according to their ecology and habitat.

4.5 - Adult Argiope bruennichi radii variation

The number of radial threads in the webs of adult A. bruennichi varied significantly over time, whereas juvenile radial threads did not significantly vary. This may be due to the general increase in the volume and the strength of the silk in adult spiders143, 64, which reduces the need for consistent radial thread construction to structurally support the web144. The fact that some webs would have been more frequently destroyed by prey impact could have influenced variation in radii number. Radial threads provide the majority of structural support in the web144and due to the difference in web building and web destruction frequency between individuals, perhaps spiders that experienced more web damage constructed more radial threads, whereas infrequent web-builders added fewer radii to their webs. Previous studies on the effect of web damage on web-building have found spiders that experience a lot of web damage tend to travel greater distances in search of new web building sites when compared to spiders that did not experience any web damage145, 146. Furthermore, web damage is highly energetically costly for spiders due to a loss of silk and effectiveness of the prey trap147. The lack of web building performed by some individual A. bruennichi may simply be due to the conservation of silk in an environment where web damage by prey was frequent. Alternatively, energetic strain of prey impact rates on webs may have been influential. One prey item was placed in the web every time a new web was built, but webs were rebuilt at different frequencies between individual spiders as some adults built up to seven webs within the same time frame as other individuals that only built three to four webs (the first three experimental webs from each individual adult were used for analysis). Therefore, it could also be the number of prey impact events on the webs of adult A. bruennichi that caused radii number to significantly vary over time, as spiders that experienced high rates of prey impact would, in theory, increase radii number over time to cope with the higher kinetic energy strain on the web57.

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However, a larger sample than six individual adults would have shown more representative results. Furthermore, prey was placed in the webs of spiders and was not entered into the webs with force, which would presumably make prey impact on the web an unlikely influence. Further investigation into radial thread construction in the subsequent webs of spiders that have experienced frequent significant web damage is needed to understand the importance of radii as a supportive aspect of the orb web in more detail. Further research on this topic may also improve knowledge on the web-building decisions made by spiders in an environment where damage to a valuable resource is frequent.

4.6 - Biotic factors more influential on web construction?

4.6.1 - Effect of food intake

In the experiment to investigate web building variation under constant abiotic conditions in A.bruennichi and Zygiella x-notata, keeping biotic conditions consistent throughout the course of web building was more difficult than keeping abiotic conditions consistent. The cephalothorax widths (mm) did not vary much within each group (adult A. bruennichi CV=10.98%, juvenile A. bruennichi CV=24.48%, Zygiella x-notata CV=10.44%), but the mass (mg) of each spider was more variable within each group (adult A. bruennichi CV=31%, juvenile A. bruennichi CV=69%, Zygiella x-notata CV=35%). Adult A. bruennichi were consistently fed on various Diptera species of similar size (10-15mm body length) and juvenile A. bruennichi and Z. x-notata were consistently fed Drosophila. However, spiders were only fed when they had built an orb web and some individuals built more frequently in the frame set-up than others, resulting in an irregularity of food intake among individuals.

Orb weavers have been known to increase the frequency of their web building and to increase variability in mesh and capture area construction in response to decreased prey capture, while showing a reduction in web building frequency and an increase in capture area over time when prey availability is increased148. This may relate to the significant variation in juvenile A. bruennichi upper mesh spacing, as these spiders were collected from the field and may have

56 had different foraging success in the wild before they were collected. Perhaps juvenile A. bruennichi with low foraging success prior to collection exhibited high variability in mesh spacing. However, this does not provide a potential explanation for left, right and lower mesh remaining consistent throughout the experiment. In addition, spiders that are in better nutritional condition have been shown to use less silk in their web building25, so perhaps radial thread investment was variable between spiders of differing nutritional states, which could have caused the significant variation in radii number over time in adult A. bruennichi webs. Again, the sample size of adult A.bruennichi was small (six individuals) and a larger sample may have shown different results. Also, this does not explain the overall consistency in other web characteristics shown by adults under constant abiotic conditions and the total consistency in web building by adult Z. x-notata. Some of the adult A. bruennichi may have had more foraging success and had obtained more nutrients than other individuals before they were collected from the field, which could be indicated in the variation in mass, which was high among the three sample groups.

4.6.2 - Prey type

Prey characteristics have been shown to influence web building with convincing supporting evidence15, 18. A more recent study observed Nephila pilipes investing more silk in a large capture area, more radial threads and longer spiral threads when fed on live Diptera compared to being fed on crickets, which triggers a reduction in radii number, radii length and enlarged mesh spacing to create a stiffer, more tense web that would be more ideal for catching larger heavier prey with a greater kinetic energy output149. In this study, all spiders were fed the same type of prey throughout their web building time apart from slight differences in prey offered to adult A. bruennichi, which may explain the lack of variation and the mostly consistent building by Z. x-notata in light and dark conditions. As mentioned before, prey was placed in the webs of spiders in this study rather than entered with force, which would presumably have a low impact on the web and a low kinetic energy output. As previously mentioned, the presence of visually acute predators can also be influential on an orb weaver’s web construction, but this was not present in this study22, 23. However, there are contradicting data, as orb weaver species exposed to Odonata and Hymenoptera prey in two separate treatments showed no changes in

57 mesh spacing in their webs, although one of the species, Larinoides cornutus, delayed its web building in the presence of the potentially dangerous Hympentoperan prey150.

4.6.3 - Prey size

The Dipteran prey size that was available for adult A. bruennichi ranged from 10-15mm in body length and although this seems like a small size range, to the spiders it may be a large enough range to influence the number of radial threads that were constructed. Larger prey tends to produce a larger kinetic energy output151, which would require more radii to increase dissipation of this energy. Perhaps only offering prey that was a more consistent size would have shown less variation in radii number between individuals. Spiders that have less frequently eaten are also known to spread their silk resources more sparingly over a web with a larger area, where radii number is reduced and mesh spacing is increased. This web design may potentially aim for the interception of larger prey, while smaller prey pass through the web, in times of hunger while investing less energy in silk152. Various studies have investigated the effect of prey type on web building15, 18, but future research should investigate how small/large the range of prey size captured by spiders must be to cause significant variation in web building over time. This will also provide valuable information on how consistent prey size must be kept in studies that are aiming to test the effects of other treatments on web building.

4.6.4 - Hydration

Intake of water is considered to be vitally important for producing silk153 and spiders need to drink loose water as well as obtaining water from their prey154. Water has been observed collecting in stabilimenta in the webs of Argiope and these spiders actively search for water on these web decorations155. The webs of adult A. bruennichi and Z. x-notata were sprayed with water at the same time as feeding. Again, there was likely to be an imbalance of hydration levels between spiders due to irregular web building, with hindered silk production in infrequently hydrated spiders.

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4.6.5 - Effect of aging

Aging can negatively impact the performance of silk glands, causing older spiders that are coming to the end of their life to invest less energy in constructing the orb web156, 157. Although all adult A. bruennichi in this study were collected as adult females, they may have slightly varied in their time of maturation in the field and this information was unknown. This may be reflected in the significant variation in radii number constructed by adult A. bruennichi over time, as the older spiders may have invested less in radial threads due to energy constraints and hindered performance of the major ampullate silk glands. However, adult A. bruennichi were collected at the same time of year, mostly in late July, to reduce the chance of significant age differences.

4.6.6 - Pollen consumption

A. bruennichi webs were dusted with pollen in order to indicate a newly built web when a pollen-free web had been constructed. Pollen ingestion by young Araneus diamematus has been shown to double the lifespan and significantly increase web building frequency when compared to spiders that were fed on fungi or were starved, suggesting that pollen is a major source of nutrients for juvenile orb weavers158. As webs among juvenile A. bruennichi were not always built in synchronisation, some spiders would have likely ingested more pollen than others when recycling and rebuilding the web. This may have cause subsequent web-building to be more frequent in some individuals and could have possibly influenced the significant variation in upper mesh over time.

4.6.7 - Effect of reproduction on web construction

Although this study did not investigate the effect of gravidness on web building, visually it appears that there could be a reduction of energy investment in the webs of three individual Z. x-notata (spiders 30, 72 and 73) that produced egg sacs during the light intensity experiment. The webs appeared to become smaller and more deformed in the build up to the day when the egg sacs were laid and appeared to contain a lower density of glue droplets on the flagelliform

59 spirals after the day of egg sac production. This was particularly clear to see in the web building of spider 73 (Images 4.1—4.4). Capture area and total thread length has been previously shown to decrease over the five webs prior to egg sac production, becoming most reduced the day before eggs were laid13. These previous observations also show that after egg sac production, mesh height stayed larger while overall thread length gradually increased over five subsequent webs, indicating that investment in the web is reduced in preparation for egg sac production13. However, Z. x-notata web building did not significantly change at all throughout the light intensity experiment.

Image 4.1 – Normal web constructed by Z. x-notata 73 the day before the egg sac was laid.

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Image 4.2 – Reduced and deformed web constructed when egg sac was found in the enclosure.

Image 4.3 – Web lacking flagelliform glue droplets constructed after the day of egg sac production. The web constructed the day after looked similar.

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Image 4.4 – Third web constructed since egg sac was laid. The web has returned to its normal state, with increased visibility due to more glue droplets on flagelliform silk.

These images appear to show reduced investment in flagelliform silk (sticky spirals) on the day and the two subsequent days after the egg sac was produced. Perhaps if future studies focused on the webs of a larger sample of gravid Z. x-notata individuals prior to production of egg sacs under constant conditions, variation patterns in web aspects would be more obvious. It is also important for subsequent research to observe changes in web building post egg production to see if web geometry consistently returns to an original state or is permanently affected by egg- laying.

4.7 - Light intensity experiment (Zygiella x-notata)

Reduction in web area in high light intensities, where insects tend to be more active, has been observed previously36. Building smaller webs in bright conditions may also have an antipredator function, to avoid prolonged periods of activity and exposure to predators42. However, again, results from this study show the contrary as there were mostly no significant

62 differences in the web geometry of Z. x-notata webs in lighter and darker conditions in the light intensity experiment.

However, the 14 Z. x-notata did build significantly larger lower mesh spacing in brighter conditions than they did in darker conditions. The reason for this may relate to silk investment and prey activity. Presumably, silk investment would be reduced when Z. x-notata built webs with larger mesh spacing in the bright condition16, 17. This could be due to prey tending to be more active in brighter conditions100, 101 as spiders can afford to invest less energy in the web due to the chance of catching prey being higher in a brighter environment. Why change just lower mesh spacing? Referring back to the ‘top/bottom’ asymmetry hypothesis, it is more energy efficient for orb weaving spiders that build webs vertically to capture prey in the lower portion of the web. This is so that the spider can travel downwards with gravity to capture the prey, rather than against gravity129. This is thought to be why many orb weavers show increased capture area below the hub117, 118. Z. x-notata may have reduced lower mesh spacing in the dark condition to prevent smaller prey passing through the web and so that, importantly, prey of almost any shape or size can be caught in conditions when prey is more scarce15. Potentially, these spiders could be using light intensity as a proxy for predicting prey activity, but as silk investment was not measured and quantified it is not possible to come to this conclusion with confidence. However, it does provide an opportunity for future research to focus not only on web geometry differences in different light intensities, but also silk investment and energy expenditure. It is also important to note that this difference in lower mesh spacing in the light and dark condition is only marginally significant (p=0.048) and a larger sample of spiders may produce different results.

Z. x-notata, from personal observation, tend to occupy the hub at night and are more active at this time, while they use their silken retreat during the daylight hours. In fact, many of the individuals collected in the field were found at night on their webs. They also tend to rebuild their webs in the early hours of morning, when there is minimal light. Perhaps night time temperature and humidity are more influential on this species’ web building, as they would be foraging/constructing webs at this time. However, it appears that day time light intensity does not influence the majority of web building of Z. x-notata, apart from lower mesh spacing potentially. It would be ideal if impending research investigated the effect of varying

63 temperature and humidity at night on the web building of Z. x-notata. This may provide the spiders with more environmental cues that would influence web construction decisions when the spider is more active and closer to the time of web rebuilding.

It would have been ideal to investigate the effect of different light intensities on the web building of Argiope bruennichi, especially with the stabilimenta aspect. However due to the seasonality of this species and the time available, this was not possible. I would encourage future research into the effect of light intensity on orb web structure to focus on Argiope species as well.

4.8 - Hierarchy of variation

Previous studies have mostly focused on the influence of a changing environment on the construction of orb webs by spiders, but there is a distinct lack of focus on how web geometry varies over time in a constant environment. In this study, there was low variation within the web building of individuals over each day from all sample groups (low CV values), although the CV values for each individual Z. x-notata differed greatly (Tables 3.2, 3.5 & 3.8). Variation in geometry of webs among all individual spiders on each day was greater (Tables 3.3, 3.6 & 3.9), but the CV values on each day are more similar to each other. Web variation between different individuals makes sense in regard to the previously mentioned factors that naturally differ from one individual to the next, such as age, individual condition and web building experience. Age may be a contributing factor to the web building variation within and between individuals over time in adult spiders as the production of silk generally decreases as time until natural death decreases156, 157. This unclear trend in response to abiotic factors may be due to the biotic environment having a stronger influence on web building behaviour.

Referring back to the ‘size limitation hypothesis’, it may be that variation within individual A. bruennichi juveniles is due to ‘errors’ in web building while the central nervous system is still developing140, but this does not provide an explanation for a similar pattern in the webs of adult. A. bruennichi and Zygiella x-notata. Further research is needed to explore web building

64 variation within individuals in a constant environment to generate hypotheses on the adaptive function of changing web structure frequently over time and what mechanisms direct this variation.

4.9 - Lack of stabilimenta

Stabilimenta only occurred 16 times out of the 88 webs built by all adult and juvenile A. bruennichi and the lack of these web decorations is unclear. However, this could be explained by findings in previous studies on stabilimenta. Wind is one of the main abiotic factors that would occur in nature, but it was not taken into consideration in this study due to the absence of wind in the laboratory set up. Orb weaving spiders have been observed enhancing stabilimenta as environmental wind speed increases159. In regard to the theory of a signalling function of stabilimenta, this could possibly be explained by strong winds increasing the likelihood of birds contacting the web, making a conspicuous signal on the web more necessary to avoid web damage, or it could be the wind generated by birds flying close that triggers an antipredator stabilimenta-building response160. The area of stabilimenta has also been shown to increase in response to airborne vibrational stimuli that mimics the approach of a predatory Hymenopteran/Dipteran, supporting an antipredator function161. Perhaps the spiders in this study would have built stabilimenta more frequently if exposed to wind or other airborne stimuli in the environment, which would occur in the wild.

The physical condition of a spider has been considered a key factor in determining stabilimenta construction. Body mass and abdomen length have been shown to positively correlate with stabilimenta area, but smaller, lighter spiders are more likely to choose to add decorations in the first place162. This is consistent with observations in this study, as the presence of decorations was more frequent in the webs of juvenile A. bruennichi (12 out of the total 16 stabilimenta webs recorded). The general lack of stabilimenta may be due to reduced food consumption in the lab (one prey item per newly built web), as web-building spiders in the field would capture more prey items during peak insect activity163 and some of these prey items may be ~200% of the spider’s body size164, although optimal prey length usually ranges from 50% - 80% of the spider’s body length in most species165. Therefore, spiders in the field are expected

65 to be heavier, longer in body length and better nourished than in the lab. A. bruennichi in this study rarely built webs in synchronisation and webs were built at different frequencies between individuals. This combination of reduced food consumption and varying web building frequency may be reflected by the irregularity of stabilimenta construction. However, the decision of stabilimenta construction by healthy spiders may be based on other factors as stabilimenta only accounts for approximately 10% of the dry weight of the web, which would presumably be energetically inexpensive to build166.

Stabilimenta construction may also be influenced by the abundance of prey in the environment. Positive associations have been made between stabilimenta building of Argiope argentata and the increased abundance of its main prey, stingless bees167. The lack of stabilimenta building by A. bruennichi in this study may due to the lack of prey in the spiders’ environment within the laboratory frame set up. Spiders only experienced the presence of prey when flies were placed into their webs. In regard to the prey attraction theory, perhaps detection of prey abundance in the environment, before it is captured, is a key trigger for web decoration- building.

4.10 - Preliminary observations of stabilimenta

In a preliminary experiment to test to effect of temperature on the web building of Argiope bruennichi, the presence of stabilimenta was noticeably different between the cooler control condition (day: 157.3µmol m-² light intensity, 20°C and 40% RH. Night: 16°C, 40% RH and total darkness. 15h light/ 9h darkness) and the warmer experimental condition (day: 77.3µmol m-2, light intensity 25°C and 40% RH, 15h light/ 9h darkness) with stabilimenta occurring 16 times in the control condition (97 webs built by 6 sdults and 10 juveniles, with stabilimenta occurring 12 times in juvenile webs) and only once in the experimental condition (41 webs built by 3 adults and 6 juveniles with stabilimenta occurring once in a juvenile web). However, due to difficulties keeping the light intensity equal in each condition due to faults in the chambers, it is not possible to determine whether it was temperature or light intensity triggering the building of stabilimenta. There were also fewer and different individual spiders in the

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experimental condition, meaning that the populations used were not independent or paired in this preliminary experiment. That said, this does provide a platform for further research into the affect that light intensity and/or temperature has on stabilimenta building decisions by Argiope bruennichi and the ontogenetic influence on stabilimenta building.

4.11 - Conclusion

The aim of this study was to investigate the influence of change in environmental light intensity on the web building behaviour of an orb weaver and to show the degree of web building variation under constant abiotic conditions. It was predicted that Z. x-notata would enhance the prey capture aspects of the orb web in lower light intensities, as in theory, insect activity would be lower in these conditions. Web building variation by Z. x-notata and A. bruennichi was expected to be insignificant in a constant abiotic environment due to the lack of environmental cues that could potentially trigger a change in web building behaviour.

In conclusion, radii number in the webs of adult A. bruennichi and upper mesh spacing in the webs of juvenile A. bruennichi significantly vary over time in a constant abiotic environment. However, the majority of web parameters remained consistent over time. Upper mesh variation over time in juveniles may be influenced by the ‘top/down asymmetry’ hypothesis, spatial constraints of the frames and/or ontogeny. Radii variation over time may be influenced by slight variations over time in prey size, web building frequency, feeding, drinking and web damage by prey. However, the exact cause of these variations remains unclear and further research is required to isolate each of these potential effects in order to observe their impact on web building in A. bruennichi.

There have not been many previous studies that have investigated web building variation under constant abiotic conditions, but the existing studies have shown that building variation in a constant environment can be significantly variable98. Here, the results mostly show the opposite, with an overall lack of variation in the webs of A. bruennichi apart from radii in adult webs and upper mesh in juvenile webs, and no significant variation in the webs of Z. x-notata. In general, individual spiders from both species vary in their web building over time (shown

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by coefficient of variation within individuals), but a population of spiders shows no overall trend over time. This indicates that web building variation within individuals could be random, as the results could possibly suggest that differences in web building among a population are not influenced by the abiotic environment, nor does a population vary the majority of web aspects in a constant environment. However, further investigation is required to support this.

There were almost no differences in the geometry of Z. x-notata webs in both treatments in the light intensity experiment, which may be due this species’ habit of being more active at night and less sensitive to day time light intensities. However, lower mesh spacing was marginally significantly reduced in the dark condition, which may relate to lower prey abundance in a darker environment and investment in silk production. Further investigation on the effect of night time abiotic factors, such as humidity and temperature, on Z. x-notata web building may be more applicable to this species. However, it could also be that the biotic environment is more influential on web building than the abiotic environment, which is supported by findings from various studies in the literature13, 15, 18, 22, 23, 25.

These findings have enhanced the limited understanding of adaptive orb web construction in response to changing abiotic factors by an orb weaver, which has proved to be mostly lacking. Variation in the fine details of the orb web over time in a constant abiotic environment is shown to be totally absent, filling important gaps in the literature on this rarely studied topic.

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Appendix

Table A1 – Total number of webs built by juvenile A. bruennichi, adult A. bruennichi and Zygiella x-notata over time. Numbered green boxes represent number of webs built by each spider. The first four webs built by each juvenile A. bruennichi were used for analysis. The first three webs built by adult A. bruennichi and Z. xnotata were used for analysis.

Spider Juvenile A. bruennichi 1 1 2 3 4 5 6 7

2 1 2 3 4 5 6

3 1 2 3 4 5 6

4 1 2 3 4 5 6

5 1 2 3 4 5 6

6 1 2 3 4 5 6

7 1 2 3 4 5

23 1 2 3 4

15 1 2 3 4 5 6

25 1 2 3 4

Adult A. bruennichi

22 1 2 3 4 5 6

17 1 2 3 4 5 30 1 2 3 4 5 6 7 32 1 2 3 4 5 6 7

19 1 2 3 4

16 1 2 3

Z. x-notata

40 1 2 3 4

34 1 2 3 4 5 6 30 1 2 3 4 5 6 7 8

32 1 2 3 4 5

13 1 2 3 4 5

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17 1 2 3 4 5

24 1 2 3 4 5

31 1 2 3

33 1 2 3

42 1 2 3

66 1 2 3

Figure A1 - Considerable left mesh spacing variation within each individual Z. x-notata over a 3-day period. Each colour represents an individual spider.

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Figure A2 – Left mesh spacing variation between adult Z. x-notata on each of the 3 days is less considerable than variation within individuals.

Figure A3 - Considerable right mesh spacing variation within individual adult Z. x-notata over a 3-day period. Each colour represents an individual spider.

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Figure A4 - Right mesh spacing variation between adult Z. x-notata on each of the 3 days is less considerable than variation within individuals.

Figure A5 - Considerable lower mesh spacing variation within individual adult Z. x-notata over a 3-day period. Each colour represents an individual spider.

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Figure A6 - Lower mesh spacing variation between adult Z. x-notata on each of the 3 days is less considerable than variation within individuals.

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