1 Impact of Relative Humidity on the Biology of Pardosa Milvina Hentz

Impact of Relative Humidity on the Biology of Pardosa milvina Hentz, 1844 (Araneae: Lycosidae)

A Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Ryan D. Bell, B.S.

Graduate Program in Entomology

The Ohio State University

2009

Thesis Committee:

Dr. Glen R. Needham, Advisor

Dr. David J. Horn

Dr. Richard A. Bradley

Ryan D. Bell

2009

Abstract

Pardosa milvina is a small wolf spider commonly associated with agricultural ecosystems. P. milvina produces dragline silk that is attached to the substrate over which it moves, but is not used in capturing prey. The effect relative humidity on P. milvina behavior and biology was examined through a series of experiments. The water balance constraints of P. milvina were studied to determine its body water content and its water loss rate at 0% RH. The calculated water loss rate is comparable to that of other terrestrial arthropods, and body water content was similar to other Pardosa spp. To examine the degree to which prey items are utilized as a water source, a study was conducted to determine if dehydrated spiders were more likely to take prey than hydrated spiders of comparable satiation levels. The individuals tested did not show an increase in prey taking when under water stress, as no spiders in either treatment took prey.

Although they did not take prey, the dehydrated spiders regained a significantly greater mass when presented with water, indicating that free-standing water sources are preferred over prey if the spider is not hungry.

The effect of relative humidity on silk deposition was examined, which necessitated the development of a technique for visualizing the silk. A difference in silk production between spiders maintained at different relative humidity levels was not found. Although there was no difference between relative humidity treatments, an

analysis of a subset of individuals by mating status did reveal a difference in silk deposition between mated and virgin females. Virgin females deposited significantly more silk than mated spiders.

iii

Dedication

To Chrissy Joy Bell, for everything she does

Acknowledgements

I would like to thank my advisor, Glen Needham, as well as my committee members Dave Horn and Rich Bradley for their guidance throughout this study. I would like to thank George Keeney and the staff of the OSU Insectary for their assistance in setting up prey cultures. I also thank Josh Benoit for his assistance and input and Justin

Whitaker for collecting help.

Vita

July 14, 1983………………………………Born – Towanda, Pennsylvania

2005……………………………………….B.S. Biology, Susquehanna University Selinsgrove, Pennsylvania

2005-present………………………………Graduate Teaching Associate, The Ohio State University, Columbus, Ohio

Publications

Bell, Ryan, A.L. Rypstra, & M.H. Persons. 2006. The effect of predator hunger on chemically-mediated antipredator responses and survival in the wolf spider Pardosa milvina (Araneae: Lycosidae). Ethology. 112:903-910.

Schonewolf, K.W., R. Bell, A.L. Rypstra, & M.H. Persons. 2006. Field evidence of an airborne enemy-avoidance kairomone in wolf spiders. Journal of Chemcial Ecology. 32:1565-1576.

Fields of Study

Major Field: Entomology

Table of Contents

Abstract……………………………………………………………...... ii

Dedication……………………………………………………………………. iv

Acknowledgements……………...... v

Vita………………………………………………………………………...... vi

List of Tables…………………………………………………………………. viii

List of Figures………………………………………………………………… ix

Chapter 1: Characteristics of Water Loss and Gain in Pardosa milvina…….. 1

Chapter 2: The Effects of Relative Humidity on Silk Deposition……………. 25

References……………………………………………………………………. 42

vii

List of Tables

Table 1.1. Water balance characteristics of P. milvina ………………………. 23

Table 1.2. Percent water changes……………………………………………... 24

viii

List of Figures

Figure 1.1. Water loss regressions for P. milvina …………………………… 22

Figure 2.1. Mean percent body mass (mg) lost ……………………………… 40

Figure 2.2. Mated vs. virgin silk indices…………………………………….. 41

Chapter 1: Characteristics of Water Loss and Gain in Pardosa milvina

ABSTRACT. Pardosa milvina is a small wolf spider commonly associated with agricultural ecosystems. The water balance constraints of P. milvina were studied to determine its body water content and its water loss rate at 0% RH. The calculated water loss rate is comparable to that of other terrestrial arthropods, and body water content was similar to other Pardosa spp. To examine the degree to which prey items are utilized as a water source, a study was conducted to determine if dehydrated spiders were more likely to take prey than hydrated spiders of comparable satiation levels. The individuals tested did not show an increase in prey taking when under water stress, as no spiders in either treatment took prey. Although they did not take prey, the dehydrated spiders regained a significantly greater mass when presented with water, indicating that free-standing water sources are preferred over prey if the spider is not hungry.

INTRODUCTION

The ability to regulate internal water balance is crucial for the survival of all organisms. Terrestrial arthropods must contend with the challenges of small size, exposure to varying temperature and humidity extremes while allowing for gas cxchange across exoskeleton. Many studies have looked at the effects of relative humidity on survival and the behavioral and physiological ways in which organisms mitigate these stresses (Wharton 1985, Hadley 1994). One of the key factors influencing terrestrial

arthropod water balance is their diminutive size, which results in a greater surface area to volume ratio compared to most vertebrates. A large surface area and small water volume places the arthropod in jeopardy due to evaporative water loss unless certain fundamental behavioral and physiological attributes are in play (Hadley 1994). Being small has its positives as well. For example, this may allow for more effective use of more optimal microhabitats of temperature and relative humidity, which can mitigate evaporative water loss (Hadley 1994). The epicuticle of the exoskeleton serves as the main barrier to passive movement of water in and out of the arthropod body. Studying the cuticle water permeability properties can provide important clues about the desiccation sensitivity of specific spider species.

To maintain water balance, the individual must match water loss with water intake or risk dehydration. Learning how much water can be lost before they become immobilized or moribund is crucial information to characterizing a spider’s water balance physiology. Body water activity (aw) is a term that is used to quantify the mole ratio of water to solutes in the arthropod. The aw will normally be around 0.99aw, corresponding to 99% water molecules (Edney 1977), while the outside environment will generally be much lower. The transpiration of water through the cuticle represents a constant loss of water that is dependent upon cuticle permeability and temperature (Hadley 1994). Even with a loss of half the body water the aw will remain near 0.99. This constant rate, which characterizes the water-loss properties of an individual, can be gravimetrically

determined by taking periodic measurements of that individual kept near 0 av or 0% humidity (RH). Vapor activity (av) is determined by dividing RH by 100. When kept in this condition at constant temperature there is no passive vapor sorption to confound the gravimetric determination. Passive movement of water vapor into an arthropod is proportional to the ambient av.

In addition to transpiration through the cuticle, there is also water lost during respiratory gas exchange. Water can also be lost via excretions or secretions. Water loss has been associated with leg grooming and glandular secretions used in short term evaporative cooling (Pulz 1987). Spider silk deposition is an additional avenue of water loss not common among other arthropods.

Water gain can come in the form of drinking, eating, metabolic water production, and active vapor uptake. Food contains some moisture, which can be an important water source for some species. Metabolic water refers to water molecules that are produced from chemical reactions, especially respiration, and active water uptake is the ability of an organism to extract water vapor from the air (Machin 1976; Gaede & Knülle 1997).

As a group, spiders have received little attention from a water balance standpoint.

They have a humidity receptor called the tarsal organ located on the tarsus of each leg, which contributes information that likely influences microhabitat selection (Barth 2002;

Ehn and Tichy 1994; Foelix 1996). Many species, including those of the Lycosidae, utilize both tubular tracheae and book lungs, which allow gas exchange while limiting

water loss (Foelix 1996). Spiders have been known to uptake water from the soil if moisture is sufficiently available (Humphreys 1975), and would gain water from prey during feeding and drinking. As with other terrestrial arthropods, spiders gain water passively from the surrounding air via passive sorption that is proportional to the ambient relative humidity (Wharton 1985). Other arachnids, most notably ticks, extract water vapor from unsaturated air by an active process (Needham and Teel 1986, 1991; Gaede and Knülle 1997). They splay their palps while extracting water from the air (Sigal et al

1999), probably to expose vapor extracting cuticular surfaces to humid air (Needham, personal communication). The lycosid Lycosa godeffroyi was not able to gain water weight from the air at near saturated conditions (Humphreys 1975); however, Lycosa howarthi gained water weight at 90% RH (Hadley et al 1981), suggesting the existence of some mechanism for active vapor uptake.

Various studies have found that the rate of water loss is influenced by a variety of factors. Weight was shown to have an impact on water loss, with larger spiders losing more total water, but at a lower percentage per weight than smaller, lighter spiders

(Humphreys 1975). Various values have been reported for the percentage of water loss that is tolerated before death. Estimates of proportional water loss resulting in lycosid death vary between studies and species, from 16-23% for L. godeffroyi (Humphreys

1975), to 20% for Hogna radiata and Alopecosa barbipes (Parry 1954), to 23% for

Pardosa pullata (Pulz 1987). Many wolf spiders will die within few days of being removed from high relative humidity (Kaston 1965).

Pardosa milvina is an abundant wolf spider, especially in agricultural systems

(Marshall et al. 2002), found throughout the eastern United States in fields, woods and near bodies of water (Kaston 1981). This is a smaller species of Lycosidae (wolf spider), with female mass approximately 20mg and males being smaller. P. milvina is primarily diurnal, although they can occasionally be captured at night (Marshall et al. 2002). They are a highly active spider (Walker et al. 1999) and can be commonly found along the hedgerows and edges of agricultural fields (Buddle et al. 2004; personal observation). It has been proposed that P. milvina and other wolf spiders originally inhabited riparian zones, but colonized agricultural fields as they became more widespread (Marshall and

Rypstra 1999). They mature during the spring, with a population peak in mid June

(Marshall et al. 2002). Mating occurs during the summer, and spiderlings overwinter as subadults.

Within the Lycosidae there is a wide variation in the degree of sexual dimorphism. Both sexes of P. milvina have similar coloration, but adult males can be readily distinguished by the characteristically dark and bulbous pedipalps, which serve as a sperm transfer structure. Males generally have a smaller abdomen in relation to their cephalothorax, and have proportionally longer legs. A study by Devito and Formanowicz

(2003) found that wolf spider males were more likely to succumb to desiccation than

were females. Male Pardosa milvina have higher metabolic rates with mass differences taken into account, but similar activity levels compared to females, (Walker & Irwin

2006). Higher metabolic rates may influence water loss rates through water lost during respiration.

Other lycosids drink soil moisture (Parry 1954). Parry (1954) showed that the spiders Hogna radiata and Alopecosa barbipes were able to draw in water from soil capillaries even against experimentally created suction, indicating that the sucking stomach of the spider is involved in uptake. It certainly seems likely that dehydrated spiders would utilize sources of free water that might be available to them, including bodies of water of various sizes as well as dew and other water droplets. Although most studies of silk recycling have focused on the energetic constraints of web building in terms of protein consumption (Higgins et al. 2001), spiders that eat their webs may be able to regain lost water due to the hydroscopic nature of their silk. Silk consumption has not been documented in wolf spiders, and does not likely provide a meaningful source of water gain.

In this series of experiments, the objectives were to establish water balance information for Pardosa milvina and determine how relative humidity affects feeding and drinking behavior. The goals of the study were to: 1) determine water loss rates (%h-1) for P. milvina for males and females of different age classes, 2) determine percent body water content for comparison to congeners, 3) confirm whether or not P. milvina drink

from standing water, and 4) determine the role that eating and drinking play in regaining lost water mass.

Water balance has been extensively studied in other organisms (Benoit et al.

2005; Yoder et al. 1997; Needham and Teel 1991), and water balance from this lycosid species further expands the water balance literature for spiders. It was hypothesized that male P. milvina would be less desiccation tolerant and would show higher water loss rates than females. Between subadults and adults, it was hypothesized that subadults would be more susceptible to desiccation.

Percent body water content of P. milvina was expected to be similar (>70%) to that of other Pardosa species (Hadley 1994). Millot and Fontaine (1937) proposed that spiders with normal body water content above 70% would need to supplement moisture from prey with additional water sources, but that spiders below 70% may be able to get all their required water from their prey. If Pardosa milvina has similar body water content to other Pardosa species, they would be expected to need to supplement their feeding with the uptake of additional water sources (Millot and Fontaine 1937). Other wolf spiders extract water from moist soil (Parry 1954), so it was hypothesized that P. milvina would drink if presented free-standing water. The role of prey in rehydration was examined by presenting both desiccated and fully hydrated spiders with prey. Between spiders under a standardized feeding regimen prior to treatment, it was hypothesized that

spiders under desiccation stress would be more likely to take prey than fully hydrated spiders.

METHODS

Spider collection and housing.–All Pardosa milvina (Hentz 1844) were collected in Delaware and Franklin counties (Ohio) the summer of 2008. Most of the individuals were collected either near a soybean field (Delaware County) or near the margins of a retreating seasonal pool (Franklin County). Specimens were maintained in the lab until utilized in 118ml semi-transparent plastic deli cups (4.5cm high x 4.5cm base diameter and 7.5cm top diameter). Each cup had a tight-fitting transparent plastic lid. A cotton cosmetic pad was placed in the bottom and moistened with distilled water.

The cotton pad was monitored weekly and re-moistened as needed and changed if it showed signs of mold growth or excessive accumulation of prey remnants. The spiders were feed a weekly diet of 5-6 Drosophila hydei flies and maintained on a 13:11 light:dark cycle at ambient room temperature (23-25°C). The D. hydei prey items were from a starter culture obtained from the Ohio State University Insectary, and were grown on a diet of re-hydrated instant mashed potato flakes, dry non-fat milk, sugar and yeast.

Water loss.–Water loss by P. milvina was studied by placing them in a desiccation chamber at near zero percent relative humidity and taking regular mass readings until the specimen perished. Individuals were treated separately and male and females, both adult and immature were used.

The standard glass desiccation chambers had a hardware screen separating the testing chamber above from the Drierite® desiccant below. A perforated aluminum foil pouch was constructed to confine the spiders while allowing for the escape of water vapor and periodic measurements without removing the specimen from the pouch.

Aluminum foil does not absorb surface moisture, allowing for more accurate measurements. A piece of foil was cut with clean scissors to approximately 8 x 12 cm while wearing gloves. It was folded in half length-wise and the long edge folded over to form a flattened tube. One end was also folded over, but the other was left open to form a tube with a final dimension of 3 x 10 cm. Perforations were made by piercing the folded tube with an ethanol sterilized thumbtack. The holes were arranged in ten rows of five holes each. A pair of clean forceps was used to open the pouch into a cylindrical shape.

Prior to the beginning of each trial, the mass of the foil pouch was recorded, then the specimen was placed inside and the pouch was carefully crimped to confine the spider. The masses of both specimen and pouch were taken before placing it in the desiccation chamber. The spider mass could then be calculated by the difference between the full and empty pouch. Measurements were taken hourly when possible, but out of necessity some measurements were taken every other hour or longer. Many of the spiders persisted more than 24 hours, with readings being mostly taken between 900 and

1900 hours. When taking measurements, the lid of the desiccator was lifted and the

pouch containing the spider quickly removed and the lid replaced. The pouch was opened and the viability of the spider was verified. If the spider was active when the pouch was opened, it was marked alive. Any immobile spiders were gently shaken to see if they were responsive. Responsive spiders that began moving were also marked alive, and completely unresponsive spiders were considered dead. If the spider showed some movement of the legs but was unable to right itself from an upside down position, it was considered alive, but severely impaired (moribund). After assessing the specimen, the pouch was crimped closed again and placed in the balance. The mass of the pouch and spider was taken and the spider was transferred back to the desiccator until the next reading.

Any spiders determined to be dead had their mass recorded, then the spider was removed and the mass of the pouch and the spider were taken separately. Both the pouch and the spider were placed in a drying oven at 39°C to remove any residual water. The pouch and spider were dried to constant mass. At this point the spider was considered to be fully dried and the final dry mass for spider and foil was taken. Any increase in mass by the pouch from the initial mass to the final dry mass was attributed to spider silk or excretory products (feces, uric acid). This mass was added to the measured spider dry mass to obtain a total dry mass for the spider. The total dry mass (dm) was subtracted from the spider mass to calculate the water mass (m) at each time interval. A value of –k was then calculated as ln(mt/m0), where m0 is the initial water mass and mt is the water

mass at time t (Wharton 1985). Values of –k were plotted against time, and regression lines were initially plotted for each individual. The resulting data set did not conform to assumptions of normality and equal variance. A logarithmic transformation was not performed as the natural log was already used to determine the value of –k. Instead, a regression line was plotted for each of the categories of spider used (subadult and adult males, subadult and adult females). Linear regression analysis generated slopes and standard error for the regression lines. The slope of the regression x 100 gives the percent water loss per hour (%h-1).

Spider drinking.–In order to conclusively determine whether P. milvina are able to imbibe free-standing water, an experiment was conducted in which desiccated spiders were offered water that had been stained with Evans blue dye. Test spiders were desiccated as described previously. Mass of the spider was recorded both before and after desiccation to document the degree of water loss.

The test chamber was comprised of a Petri dish fitted with a sleeve made of a cylindrically folded sheet of acetate to extend the sides of the chamber. The spider was introduced to the chamber underneath an inverted empty film canister that had been swabbed with ethanol and allowed to dry. The spider was positioned to one side of the

Petri dish, and a 50µl drop of Evans blue stained water was added to the chamber directly across from the spider with a micropipette. The spider was given 1 minute to acclimate and then the film canister was lifted out of the chamber, allowing the spider to move

freely about the chamber. The spider was then observed to see if it found the dyed water and if it demonstrated any noticeable behavior that may be indicative of active drinking, namely lowering of the mouthparts into the droplet of water. To aid in observations, a video recorder was attached to a side mounted dissection scope to allow for a magnified view of the droplet while limiting disturbance. If the spider did touch the droplet with its mouthparts, it was allowed to remain in contact with the water. When the spider was no longer contacting the droplet the trial was ended and the spider removed and the mass taken to see if there was a gain due to uptake of water. The spider was then killed and dissected to determine if there was evidence of water uptake in the form of blue dye found internally.

Satiated dehydrated vs. satiated hydrated spiders.–Feeding upon prey may be an important source of water for spiders. This experiment was designed to examine whether dehydration level has an impact on the likelihood and motivation for a spider to kill and consume prey.

Experiments were set up such that satiated P. milvina (both male and female subadults) were divided into two treatment groups, one maintained at saturated humidity conditions (chamber with a distilled water reservoir), and one at desiccating conditions

(chamber with Drierite®). Prior to the beginning of the trial, all spiders were fed ad libitum for 24 hours to allow them to become fully satiated. The mass was recorded for each individual and they were placed in perforated foil pouches and placed in the

treatment chambers for ~ 20 hours. After exposure to these relative humidity levels, they were removed and the mass recorded. Each spider was placed within an alcohol sterilized 118mL deli container that had been allowed to fully dry.

To each container, four D. hydei were added and the spider and flies were left undisturbed for two hours. Before adding the flies to the container, the total mass for the four flies had been taken. After two hours the P. milvina were observed and any feeding on the flies was noted. Spider mass was again recorded, before returning the spider to the container. Each spider was presented with 20µl of distilled water that was added to the bottom of the deli container with a micropipette. After 30 minutes the spiders were removed from the container and placed within a vial lined with absorbent paper towel to remove any moisture that may have been present on the legs before taking the mass of the spider. The effects of treatment on mass lost while in the treatment chambers, and on mass gained from water were analyzed using a t-test.

RESULTS

Water loss.– The rate of water loss (%h-1) was derived from the slope of the regression line of ln(mt/m0) plotted against time (Figure 1.1). A common linear regression was used for each group because linear regressions of individual spiders did not fit normality assumptions to allow for use with an ANOVA. Multiplication of the slope by 100 gives the %h-1 value. Adult females lost water at a rate of 0.970 ± 0.05 %h-1

(Figure 1.1.A, Table 1.1) and subadult females at a rate of 0.803 ± 0.02 %h-1 (Figure

1.1.B, Table 1.1). Adult males lost water at a rate of 1.17 ± 0.05 %h-1 (Figure 1.1.C,

Table 1.1) and subadult males at a rate of 1.18 ± 0.03 %h-1 (Figure 1.1.D, Table 1.1).

Water balance characteristics were calculated for the four groups of spiders (Table 1.1), including mean initial mass, dry mass, water mass, and percent water content.

Pardosa drinking: All three dehydrated spiders showed behaviors consistent with intentional drinking and uptake of free water when presented with a droplet of dyed water. All three had a measurable loss of body mass during the desiccation portion of the trial, as well as a gain in mass after drinking from the droplet. The blue dye was found internally when the spiders were dissected verifying that water was consumed.

Desiccated vs. Hydrated: No individuals in either the desiccation or hydration treatments took prey. As expected, spiders kept near 0% RH did show a significantly greater loss in mass after being kept in desiccating conditions (Table 1.2; p = 0.003).

After being given access to free-standing water, the dehydrated spiders had a significantly higher gain in mass over the spiders in the hydrated treatment (Table 1.2; p

< 0.001).

DISCUSSION

Water loss: Water loss rates were calculated from the slopes derived from the linear regression of –k (Figure 1.1), for each of the four groups of spiders. There is a general trend of females (Figure 1.1A and 1B) being more water tight than males (Figure

1.1C and 1D), which had higher water loss rates. For females, the %h-1 for adults (Figure

1.1A) was 0.97%h-1 ± 0.05 and 0.8%h-1± 0.02 for subadults (Figure 1.1B). The %h-1 for males was very similar between adult (Figure 1.1C, 1.18%h-1 ± 0.05) and subadult males

(Figure 1.1D, 1.17%h-1 ± 0.03). These values represent higher water loss rates for males of both maturity levels than was found among females. Although analysis comparing the age and sex groups was not performed due to issues with conforming to statistical assumptions, there does appear to be a trend for higher water loss rates in males than females, regardless of whether they are adult or subadult. Survival studies on the riparian wolf spider Pirata sedentarius (DeVito and Formanowicz 2003) found that males were likewise less desiccation tolerant, which would be predicted for P. milvina based on their higher water loss rates.

The adult females had a larger mean mass (25.29 ± 0.39 mg, Table 1.1) than any other groups, which were all similar in mass (Table 1.1, subadult females 16.08 ± 1.21; adult males 13.99 ± 0.44; subadult males 14.7 ± 0.39). Although mass provides a gauge of size and surface area, it changes with the morphological differences between maturity levels and sex. Male P. milvina have smaller bodies than females, but proportionally longer legs, which results in a higher surface area to volume ratio possibly predisposing them to a higher rate of water loss.

In the adult males, the spiders were used within a few weeks of molting from subadult to adult spiders, but the adult females included some spiders that had been fully mature for longer periods of time. This may have also contributed to the greater variation

and spread in the data points used in the regression of the adult female spiders (Figure

1.1A). Older adult females may have had higher water loss rates than adult females that had only recently molted. The water loss rates for Drosophila melanogaster have been observed to increase with age (Fairbanks and Burch 1970). Other studies have shown that as ticks age, they become more permeable and lose water at a faster rate (Needham and Teel 1991, Yoder et al. 1997). Since P. milvina do not molt again after becoming adults, abrasion and loss of setae may result in an increase in transpiration (Davies and

Edney 1952) as they age. This contention needs to be tested for P. milvina. Using more individuals of a consistent age range within the maturity groups may resolve some of the variability issues that complicated comparisons between groups.

The water loss rates for P. milvina can be compared to other arthropods in which

% h-1 rates have been examined. The water loss rates for P. milvina are much higher than those reported for ticks (0.0638 – 0.323 %h-1 Needham and Teel 1991; 0.1712 – 0.2736

%h-1 females and 0.2146 – 0.3217%h-1 males, Yoder et al. 1997) and spider beetles (%h-1

< 0.06, Benoit et al. 2005) and lower than the rates for adult fleas (2.43 %h-1, Thiemann et al. 2003; 1.73 and 1.39 %h-1, Fielden et al. 2002).

Relative amounts of body fat and the mass of the cuticle can impact the mean percent body water, as both body fat and cuticle are low in water content (Edney 1977).

Differences in the amount of fat and cuticle present between life stages and sexes can influence the calculated percent body water (Edney 1977, Hadley 1994). ANOVA

analysis did not show a statistical difference in mean percent body water content (Table

1.1) across groups (p = 0.10). Although mean percent body water was slightly higher for adult males, it was not significantly higher. Data was combined for all individuals (n =

24), for a mean percent body water content for P. milvina of 69.9 ± 0.60%. This is identical to the 70% that Millot and Fontaine (1937) proposed as being the dividing line between spiders that are able to obtain all necessary water from prey and those that must supplement with additional sources. Hadley (1994) gave percent body water values for two other Pardosa species that ranged from 74 – 77%, which is slightly higher than what was found with P. milvina. It seems that the relationship between body water content and supplementing with water sources holds true for P. milvina, and based on body water content for other Pardosa species, these behaviors may be found through the genera.

Water loss management is an important adaptation for terrestrial arthropods in particular, which often have a high surface area to volume ratio. As the animal dehydrates, water may be remobilized from the hemolymph and made available to the cells to help maintain the correct osmotic balance. During the water loss study, spiders were checked and mass recorded periodically. Observations were made of spiders that were unable to move in a manner consistent with normal behavior. This included partial extension of the first pair of legs when walking, seemingly uncoordinated movement of the legs, and the inability to right itself when resting on its dorsal surface. Spiders in which impairment was noticed died during the subsequent measurement interval. There

seems to be a range of severity to the impairment of the spider as they become more dehydrated. Spiders that exhibited a lack of coordination following a period of desiccation showed an inability to fully extend the first pair of legs, resulting in the distal segments dragging as they moved forward. Ellis (1944) reported similar walking movement in tarantulas that had been kept without access to water for an extended period of time. When injected with a saline solution, the spiders were able to regain normal walking movement (Ellis 1944). The hydrostatic extension of the legs at the femoral- patellar joint and the metatarsal-tarsal joint are achieved by an increase in hemolymph pressure (Ellis 1944, Foelix 1996, Parry 1957). If there is too much of a decrease in the hemolymph volume, the spider may be unable to generate enough hydrostatic pressure to extend its appendages. Eventually, if the spider reaches a level of dehydration such that they are unable to extend their legs, it seems unlikely that they will be able to move to a more favorable location to find a source of water or capture prey.

Pardosa drinking: All spiders used in the drinking trials had been pre-desiccated.

When the vial was removed, some of the spiders found the droplet sooner than others, but they all stopped at the droplet when it was first encountered. They placed their chelicerae in contact with the water for minutes at a time before moving away. Some moved off for brief periods and returned. In these instances human disturbance may have caused this behavior. Video camera and dissecting scope set up allowed for observations of a drinking spider, which confirmed that the mouthparts and chelicerae were in contact with

the water droplet. All specimens increased in mass following their bout of drinking from the droplet and dye was found upon dissection, confirming a net uptake of water.

Desiccated vs. hydrated: Prey items provide some water, and for spiders with body water contents below 70%, this may be a sufficient moisture resource (Millot and

Fontaine 1937). Spiders that naturally maintain water content above 70% have to supplement their prey consumption from other sources (Millot and Fontaine 1937). I hypothesized that fully fed spiders under water stress would take more prey items than fully fed, hydrated individuals. Spiders were preconditioned by being held at desiccating conditions for less than 24 hours, however, none killed prey during the course of the trial.

This indicates that desiccated spiders are not more likely to take prey just to rehydrate but this kind of experiment needs to be repeated to confidently accept or reject any hypothesis regarding hydration state upon prey taking for rehydration.

Spiders from the desiccation treatment showed a significantly greater loss in total body mass (as a percent of initial body mass, Table 1.2). As expected, more water was lost by the spiders maintained near 0% RH (Drierite®) than by those near 100% RH

(distilled water). At relative humidity levels above 0%, there will be passive uptake of water molecules through the cuticle proportional to the ambient relative humidity. At subsaturated levels, this passive uptake of water vapor is usually less than the amount of water that is lost through transpiration and other water loss, resulting in a net loss of water from the individual (Wharton, 1985).

When offered free-standing water following presentation of prey, spiders exhibited behaviors associated with drinking as described previously when coming in contact with the droplet. Spiders that had been kept in the desiccating treatment regained a significantly higher percentage of their original body mass back after thirty minutes of access to the water (Table 1.2).

If taking prey is a viable strategy for P. milvina to replace lost water, it is possible that dehydrated individuals will reach some threshold at which it becomes advantageous.

If such a threshold exists, it would have to fall somewhere between the level of dehydration found in this study and the point at which there are physical limitations on the ability to take prey. In this study, no prey was taken by individuals in either treatment, but all of the dehydrated spiders gained water mass when permitted to drink.

This may be indicative of P. milvina behavior under water stress, suggesting that they may be more likely to take up water from other sources (e.g. dew, standing water) rather than from additional prey. The other aspect of this relationship between prey taking and hydration level would be the critical point at which the spider has lost too much water to be able to take prey. In addition to the hydrostatic limitations in the legs under desiccated conditions, spider feeding behavior may present another cost that would limit the likelihood of taking prey. The regurgitation and subsequent uptake of predigested prey

(Foelix 1996) constitutes an investment of body moisture by the spider (Pulz 1987), which may become more crucial under desiccation. It is possible that at some point,

hydration through free-standing water would be necessary prior to the spider being able to take prey.

The observations of the spiders used in the feeding and drinking behavioral study suggests that P. milvina is a species that needs to supplement dietary water with drinking free-standing water. The mean percent body water of 69.93 ± 0.60% is compatible with the hypothesis that 70% marks the level at which spiders will need access to water sources in addition to prey (Millot and Fontaine 1937).

Conclusions: These studies provide new information on the water balance characteristics of the wolf spider Pardosa milvina demonstrating a trend for higher water loss rates in males compared to females at two different maturity levels. Further studies with an increased sample size could be done to look at the effects of age on water loss rates by establishing treatment groups for both sexes with subadults, recently molted adults, and older adults. Dehydrated spiders were not found to be more likely to take prey for rehydration purposes, but do demonstrate a proportional increase in water gain through drinking when significantly dehydrated. This supports Millot and Fontaine’s

(1937) statement that spiders above 70% body water content will need to supplement moisture from prey with additional water sources.

Figure 1.1.Water loss rate regressions for P. milvina. The slope of the regression line represents the %h-1 water loss. A) Adult females (n = 6), %h-1 = 0.970 ± 0.05; B) Subadult females (n = 8), %h-1 = 0.803 ± 0.02; C) Adult males (n = 4), %h-1 = 1.17 ± 0.05; D) Subadult males (n = 6), %h-1 = 1.18 ± 0.03. Data was not collected overnight, producing gaps in the data for those spiders that survived more than 24 hours.

Table 1.1. Water balance characteristics of P. milvina of both sexes and different maturity levels. ANOVA analysis of mean percent body water content did not detect a statistical difference (F = 2.356, p = 0.10) among groups. Mean percent body water for all individuals 69.9 ± 0.60%.

Characteristic Adult female Subadult female Adult male Subadult male Water content f (mg) 25.29 ± 1.94 16.08 ± 1.21 13.99 ± 0.44 14.70 ± 0.39 d (mg) 7.79 ± 0.90 5.03 ± 0.38 3.77 ± 0.18 5.03 ± 0.38 m (mg) 17.50 ± 1.07 11.05 ± 0.85 10.22 ± 0.42 11.05 ± 0.85 % 69.63 ± 1.75 68.68 ± 0.52 73.02 ± 1.29 68.68 ± 0.52

Water loss %h-1 0.970 ± 0.051 0.803 ± 0.023 1.17 ± 0.049 1.18 ± 0.029 f, fresh initial mass; d, dry mass; m, water mass; %, percent body water content, %h-1, water loss rate.

Table 1.2. In dehydrating and hydrating conditions, a significant difference was found following a t-test between treatments both in the percent mass lost while under treatment conditions (p = 0.003) and the percent mass regained after being presented with free- standing water (p < 0.001). No individuals in either treatment took prey when given access.

Treatment N % mass lost % mass regained (water) 0% RH 7 18.65 ± 3.1 a 11.67 ± 1.7 a 100% RH 9 3.72 ± 0.69 b -0.13 ± 1.9 b

Chapter 2: The Effects of Relative Humidity on Silk Deposition

ABSTRACT. Pardosa milvina Hentz 1844, like other wolf spiders, produces dragline silk that is attached to the substrate over which it moves, but is not used in capturing prey. The effect of relative humidity on silk deposition was examined, which necessitated the development of a technique for visualizing the silk. A difference in silk production between spiders maintained at different relative humidity levels was not found. Although there was no difference between relative humidity treatments, an analysis of a subset of individuals by mating status did reveal a difference in silk deposition between mated and virgin females. Virgin females deposited significantly more silk than mated spiders.

INTRODUCTION

Wolf spiders do not build webs for use in prey capture; instead they leave silk draglines as they move around the environment. Male wolf spiders utilize female silk in mate finding (Roland 1984, Taylor 1998), presumably following pheromones bound to the silk. Mating usually only takes place after a period of courtship by the male in which they engage in a series of species specific behaviors. This may include palpal chemoexploration (Brown 2006) accompanied by leg waving or other visual signals as well as vibratory cues on the substrate (Hebets and Uetz 2000).

Silk is an interesting biological product that may play a role in the water balance of spiders. Silk proteins are produced in opisthosomal glands as a water soluble liquid that is extruded from the spinnerets. As it is passes through silk ducts and is extruded, the silk undergoes an ion and pH induced conformational change and becomes a solid strand (Foo et al. 2006). The lining of the duct leading from the silk gland to the spigot of the spinneret is able to reabsorb some of the water from the liquid silk. Water content of dragline silk as it emerges from the spider has been found to be 20% in the orb weaving Nephila edulis (Foo et al. 2006). Water content of silk has an important impact upon its physical properties and becomes brittle if too dry. High water content is responsible for silk stickiness among the orb weaving spiders (Foelix 1996). As the capture silk is produced, it is coated with a hydroscopic layer that attracts additional vapor from the air (Edmonds and Vollrath 1992; Higgins et al. 2001). Coating the capture silk with this hydroscopic layer saves the spider from using its own water to produces sticky silk and ensures that the silk can retain its properties even in changing conditions. Since some water is lost during silk production (Foo et al. 2006), costs associated with silk production may have an impact at low relative humidity levels where excess water loss becomes more hazardous to the spider (Vollrath et al. 1997).

Additionally, silk deposition may become more difficult for spiders that are dehydrated, as an increase in hemolymph pressure is thought to play a role in extrusion of silk from the glands (Foelix 1996). Dehydration has been shown to decrease movement in leg

joints that rely on hemolymph pressure for extension (Ellis 1944). However, it seems likely that at dehydration levels that may impair silk extrusion, the mobility of the spider would be affected as well.

It is not clear to what degree the water lost during silk production impacts the water balance of spiders. Although silk water content of 20% has been reported (Foo et al. 2006), this was only looking at one species and silk from one gland. The ampullate glands that produced the silk studied by Foo et al. (2006) in Nephila edulis are the same glands from which lycosid drag line silk is produced. There is likely a wide range of variation in water content between species and even between silk types produced by the same spider. Gravimetrically determining the water content of the silk contributed by the spider would be difficult given the relatively low mass of silk strands, as well as its hydroscopic nature. Because silk is light weight, even a 20% water contribution by the spider may not be physiologically significant under normal conditions. Although most studies of silk recycling have focused on the energetic constraints of web building in terms of protein consumption (Higgins et al. 2001), spiders that eat their webs may be able to regain lost water due to the hydroscopic nature of their silk. Silk consumption has not been documented in wolf spiders, and does not likely provide a meaningful source of water gain for the reasons discussed above.

Silk quantity is harder to assess in ground dwelling spiders than in web builders, necessitating the development of a new technique for visualizing and quantifying the

deposition of silk by wolf spiders. Silk production in orb builders can be quantified by the area of the web (Blackledge and Gillespie 2002, Vollrath et al. 1997), but this is not applicable to silk produced by wolf spiders. Because the silk is attached to the substrate, it can sometimes be hard to visualize. In this study a new technique was developed to allow for the deposition of silk on a medium that allowed for the collection and storage of the deposited silk for future analysis, as well as a method for reliably sampling the stored silk.

The question being asked was whether relative humidity has an impact on the amount of silk deposited on a substrate. Spiders have been shown to reduce web size in low humidity (Vollrath et al. 1997), but it has not been established whether this is due to water loss costs associated with silk production or due to perceived reduction in prey capture potential. Higher levels of activity are associated with greater water loss (Pulz

1987), and a reduction in activity to limit water loss would result in less silk deposition.

It was hypothesized that P. milvina would deposit less silk at lower relative humidity levels.

METHODS

Spider collection and housing.–All Pardosa milvina were collected in Delaware and Franklin counties (Ohio) the summer of 2008. Specimens were maintained in the lab until utilized in 118ml semi-transparent plastic deli cups (4.5cm high x 4.5cm base diameter and 7.5cm top diameter) with a tight fitting transparent plastic lid. A cotton

cosmetic pad was placed in the bottom of each container and moistened with distilled water to meet water requirements. The cotton pad was monitored weekly and re- moistened as needed and changed if it showed signs of mold growth or excessive accumulation of prey remnants. The spiders were feed a weekly diet of 5-6 Drosophila hydei flies and maintained on a 13:11 light:dark cycle at ambient room temperature (23-

25°C). The D. hydei prey items were from a starter culture obtained from the Ohio State

University Insectary, and were grown on a diet of re-hydrated instant mashed potato flakes, dry non-fat milk, sugar and yeast.

Silk deposition experiment.–All specimens used in silk deposition studies were adult females that were provided water and were fed ad libitum with D. hydei 48 hours prior to the beginning of the trial. After 24 hours all living prey items were removed, as well as any uneaten or partially consumed dead prey items. All trial specimens were maintained without prey, but with access to water for the remaining 24 hours prior to the beginning of the trial to standardize satiation levels before being used to deposit silk.

Silk deposition data was collected using individual collection chambers placed within a humidity chamber (40, 60, or 80% RH).

The bottoms were cut out of a 118ml deli cups and each was inverted to form the silk collection chambers. The lid served as the base of the chamber and the tapered cylinder functioned to limit spider escape and wall climbing. The top was open to the humidity chamber, allowing for vapor exchange and equilibration with the humidity

chamber. A circular disk of filter paper was cut to 6.5cm in diameter to fit the base of the collection chamber. To collect silk, small pieces of double sided tape were used to fix two circular glass cover slips measuring 1.2cm in diameter to the filter paper to ensure that they did not slide during silk deposition or handling. Taping the cover slips to the filter paper made the collected silk more stable for storage and subsequent analysis. The cover slips were placed on opposite sides and 1 cm from the edge of the filter paper to limit potential edge effects. To assemble the collection chamber, the filter paper with the cover slips was centered on the base and the top snapped down into the base.

The humidity chamber was made from a ten gallon (71cm x 41cm x 13cm) glass aquarium tank fitted with a foam gasket and a glass lid to minimize vapor exchange with the outside environment. To each tank was added a glycerol-water solution to establish the desired relative humidity level for each of the three treatments. The glycerol water solutions were made using percentage by mass, based on the percent glycerol/percent water ratios from Miner (1953). Glycerol/water solutions of 800g were made, generating relative humidity levels of 40, 60, and 80% and were poured in the bottom of the corresponding treatment tank. Wire test tube racks were used to elevate the individual silk collection chambers above the solution without limiting the surface area of the solution.

Before each trial the base and top of each collection chamber was cleaned with

75% ethyl alcohol and allowed to dry. All chamber components were handled either with

clean, gloved hands, or using a clean pair of forceps. The filter paper was cut with scissors that had been similarly sterilized with alcohol, and the double sided tape cut with a razor blade that had also been sterilized. Spider mass was recorded with an analytical balance (Mettler Toledo AX205 DeltaRange®) and the individual was placed in a plastic vial. Once the collection chamber was assembled, the spider was introduced and the chamber immediately lowered into the appropriate tank along with a data logger

(HOBO® H8 RH/Temp, The Onset Computer Corporation) to document relative humidity and temperature at fifteen minute intervals, and the lid replaced. The chamber lid was off of the tank as briefly as possible to reduce vapor exchange, which could delay humidity equilibration within the tank.

Spiders were kept in the tank for 24 hours, at which point they were removed and transferred from the collection chamber to a plastic vial to prevent any more silk deposition. The post-treatment mass was then recorded for each specimen, after which they were preserved in 75% ethanol for voucher purposes (The Ohio State University,

Acarology Collection, Museum of Biodiversity). The filter paper was removed from the silk collection chamber and the cover slips and surrounding filter paper were cut from the rest and placed in labeled containers for future analysis. Temperature and relative humidity data were downloaded from the data logger onto a computer. Mean temperature (°C ± SEM) and mean relative humidity (%RH ± SEM) were determined for each treatment.

Data collection and analysis: The cover slips were carefully detached from the filter paper and the double-sided tape removed. The cover slips were weighed and then placed on top of a hemocytometer counting chamber under a compound microscope with a camera attachment. The hemocytometer provided a background of grid-lines that were used to quantify the silk deposited in the collection chamber. The hemocytometer used the Improved Neubauer ruling pattern, which divides a 3 x 3mm area into nine 1 x 1 mm sub-regions.

Each slip was centered on the counting chamber and four digital photographs taken, one of each quadrant of the counting chamber. These four images were then merged into a composite photo for analysis. Within each grid, only the four corner sub- regions of the grid were quantified. The other five sub-regions had additional dividing grid-lines, making it more difficult to see the silk. Each of these corner sub-regions was comprised of sixteen squares. Each square was assigned a value based on the number of strands of silk it contained. The values for all 128 squares (16 squares x 4 corners x 2 grids) were summed to generate a silk index for each spider.

The silk indices were transformed by ln(x + 1) to conform to normality assumptions. The assumption of equal variance was verified with Levene’s test.

Statistical significance was tested for using a one-way ANOVA. The pre and post- treatment mass data recorded for the spiders were analyzed to examine the effects of treatment on spider mass. A one-way ANOVA was used to determine statistical

significance of percent mass lost across treatments with a Tukey post-hoc comparison of means test.

Mating status information was available for a subset of the individuals used in the silk deposition experiment. Any spiders that were captured with an egg sac or spiderlings were determined to be mated, as were any adult females that produced an egg sac while in the lab. Spiders captured as immatures that molted into adults while in the lab were known to be virgins. Females captured as adults that did not produce an egg sac prior to being used in the experiment were excluded from this analysis as their mating status could not be ascertained. A comparison of the silk index data between mated and virgin females conformed to assumptions of normality, and was analyzed using a t-test.

RESULTS

Silk deposition.– Silk index data fit normality assumptions after ln(x + 1) transformation and was analyzed using one-way ANOVA. A significant difference between treatments was not found (p = 0.146). The null hypothesis of equal mean silk index between treatments could not be rejected.

Data from the HOBO data loggers confirmed that the conditions within the test chambers were close to the intended experimental conditions. The relative humidity in the 40% RH treatment was slightly lower, with a mean RH of 36.38 % ± 0.07 at 23.95°C

± 0.08. The 60% chamber had a mean RH of 58.57 % ± 0.07 at 23.15°C ± 0.04, and the

80% chamber had a mean RH of 80.50 % ± 0.2 at 23.22°C ± 0.05.

Percent mass loss across treatments: Mass loss between relative humidity treatments in the silk deposition study was analyzed. Mass loss was calculated from initial and final masses during the 24-hour trial. The difference was divided by the initial mass to get the percent mass difference for each individual. A probability plot confirmed that the percent mass data conformed to a normal distribution and Levene’s test showed equal variance between the treatments. A one-way ANOVA found a statistically significant difference for the percent mass lost between the treatments (Figure 1) (f =

8.25, p-value = 0.001). Pair-wise comparisons were made by calculating a least significant difference (Tukey LSD) value for mean percent loss at a 0.05 alpha level.

Statistical significance was found in the mean percent mass lost between 40 and 60% RH and 40 and 80% RH treatments, but not between 60 and 80% RH.

Mated vs. virgin: A comparison of silk indices for mated and virgin spiders was performed to determine if there was a treatment effect due to mating status. The silk index data for mated and virgin individuals fit assumptions of normal distribution and equal variance. Virgin females deposited more silk than mated females, and t-test analysis of silk index values supported rejection of a null hypothesis of no difference between females of different mating status. A statistical significance was found between mated (108.3 ± 40 SE) and virgin (273.7 ± 23 SE) spider silk indices (p-value = 0.024)

(Figure 2).

DISCUSSION

Silk deposition: Lycosid spiders lay down silk as they move about. Silk deposition is a function of the quantity and the distribution of silk by the individual lycosid spider. It was difficult to differentiate between the effects of quantity and distribution on the silk index counts generated because of how the data was collected.

Spiders may have had a higher level of activity shortly after being introduced the silk collection chambers as a result of handling and exploration of new surroundings. An artificial increase in activity could have contributed to additional deposition of silk draglines. The relative humidity condition within the chambers also takes some time to re-establish using the glycerol-water solution. During this time there may be movement by the spider that is not representative of their movement under the experimental conditions. Due to the design of the collection equipment, it would not be possible to acclimate the spiders to experimental conditions before collecting data. Another way of examining spider behavior under varying relative humidity would be with video tracking.

Although this would not directly answer the question of silk deposition, it would allow for acclimating the spiders to their enclosures and provide data on locomotion, which silk deposition is dependant on.

Some individuals crawled under the filter paper if it did not fit tightly to the walls of the chamber. Although they were not included in the silk analysis, this may indicate

that individuals were attempting to find a more suitable microhabitat within the silk collection chamber.

Other factors in the life history and phenology of the individual spiders may have a large impact upon their silk production. Field caught individuals may more accurately represent the natural behaviors of the population, but there is usually higher variation between individuals, and there is no control over other factors within the individual’s past that may affect results. On the other hand, completely lab raised individuals allow for more control of phenology and development, but may produce lab artifacts that are not representative of the natural population. In these experiments, the specimens were collected from wild populations and maintained in the lab, some for longer periods than others. This may have contributed to additional variation among individuals.

Percent mass lost across treatment: ANOVA analysis and Tukey LSD pair-wise comparison between treatments of the mean mass lost found a statistically significant difference between treatments (Figure 1). Spiders kept at 40% RH lost a significantly higher mean percent mass (15.48 ± 1.08 %) during the silk deposition trial compared to both the 60 (11.87 ± 1.22 %) and 80% RH (9.26 ± 0.95 %) treatments. However, there was not a significant difference in percent mass lost at 60 and 80% RH. Although the difference in mass loss was statistically significant, it is not known whether the losses noted during this experiment were physiologically significant in terms of reduced movement or silk production.

Mated vs. Virgin: The comparison of silk index between mated and virgin females

(Figure 2) indicates that silk deposition is influenced by mating status. Virgin spiders deposited more silk (108.3± 40) than mated specimens (273.7± 23), which was statistically significant following a t-test (p-value = 0.024). The mean mass of the mated spiders was higher than that for the virgins, so it does not seem to be a case of larger spiders producing more silk. Out of the spiders used in the silk deposition study, the mating status was known only for seven individuals (mated n = 4; virgin n = 3).

Increasing the sample size and controlling for the length of time from mating to the experiment would make this study more robust.

Although unmated spiders may produce egg sacs (Montgomery 1907, Edwards et al. 2003) they are almost invariably sterile. Although rare, parthenogenesis, or the development of offspring from unfertilized eggs, does occur in spiders (Edwards et al.

2003, Phillips 1903). In lycosids, production of egg sacs with unfertilized eggs is uncommon, and does not seem to produce viable embryos (Montgomery 1907). The reason for an unmated spider to produce a sterile egg sac is not clear, but abundant nutrients resulting from a lab diet may mitigate energetic costs that would normally be associated with egg sac production. If the eggs are not viable, or otherwise damaged, the female may be more likely to drop or eat the egg sac (Kaston 1965). It was assumed that the production of an egg sac in the silk deposition study was an indicator that mating had occurred.

Since silk and silk-related pheromones serve a role in mating (Foelix 1996), it is possible that there is a selective pressure on unmated females to deposit more silk to facilitate finding a suitable mate, a selection pressure that decreases following copulation.

Although females will occasionally accept more than one male and copulate multiple times (Montgomery 1903), the benefits decrease with each additional mating beyond an optimal rate (Arnqvist and Nilsson 2000). Potential benefits may include increased fitness in their offspring (García-González & Simmons 2005). Females may also cannibalize their mates resulting in a nutritive gain. Mating multiple times may potentially be costly to the female by increasing visibility to predators or incurring other costs. Even if the female does not mate with additional males, unsolicited attention from males could constitute a cost to the female. In addition to increased predation risk, there would be energetic costs associated with repelling unwanted males.

If a higher level of silk production is a function of an increased selection to find a mate, a female that has already mated and acquired the sperm needed to produce spiderlings may reduce the amount of silk deposited. If it is selective for mated females to reduce the ability of courting males to detect them, there may be subsequent changes in their silk, both quantitatively and qualitatively. There may also be a change in the pheromone composition associated with the silk between mated and unmated females, which could change male response.

Alternately, female behaviors and receptivity following mating may be due less to female choice and more to male influence through copulatory transfer of compounds that reduce the likelihood of addition mating by the female.

Other factors that could have contributed to variation between mated and virgin spiders include age and treatment effects. To more fully examine effects of mating status on silk deposition, further studies should be conducted using spiders of a standardized age and life history in consistent environmental conditions.

18 40% a 16 60% b 14

) 80% b g

m 12 % (

t s

o 10 l

s s a

m 8

t n e c

r 6 e P 4

0 Humidity treatment

Figure 2.1. Mean percent body mass (mg) lost during 24hr silk deposition by spiders (N = 12 per treatment) in 40, 60 and 80% RH. Error bars represent SE. A statistically significant difference in percent body mass lost was found with one-way ANOVA (F = 8.25; p = 0.001). Pair-wise comparison of means was done using Tukey LSD, with letter designation in the legend representing statistical significance based on a 0.05 alpha level.

Figure 2.2. Mean silk index responses for mated and virgin females. Silk index fit a normal distribution and was analyzed using t-test. A statistical significance was found between the mean silk indices for mated (108.3± 40 SE) and virgin (273.7± 23 SE) spiders, with a reported p-value = 0.024.

350 Mated Females 300 Virgin Females

x 250 e d n i

200 k l i s

n 150 a e

M 100

0 Spider mating history

References

Arnqvist G. & T. Nilsson. 2000. The evolution of polyandry: multiple mating and female fitness in insects. Animal Behaviour. 60:145-164.

Barth, F.G. 2002. A Spider’s World Senses and Behavior. Springer, Berlin.

Benoit, J.B., J.A. Yoder, E.J. Rellinger, J.T. Ark & G.D. Keeney. 2005. Prolonged maintenance of water balance by adult females of the American spider beetle, Mezium affine Boieldieu, in the absence of food and water resources. Journal of Insect Physiology. 51:565-573.

Blackledge, T.A. & R.G. Gillespie. 2002. Estimation of capture areas of spider orb webs in relation to asymmetry. Journal of Arachnology. 30:70-77.

Brown, C.A. 2006. Observations on courtship and copulation of the wolf spider Rabidosa santrita (Araneae, Lycosidae). Journal of ARachnology. 34:476-479.

Buddle, C.M., S. Higgins & A.L. Rypstra. 2004. Ground-dwelling spider assemblages inhabiting riparian forests and hedgerows in an agricultural landscape. American Midlands Naturalist. 151:15-26.

Cloudsley-Thompson, J.L. 1957. Studies in diurnal rhythms. V. Nocturnal ecology and water-relations of the British cribellate spiders of the genus Ciniflo Bl. Journal of the Linnean Society of London, Zoology. 43:134-152.

Davies, M.E. & E.B. Edney. 1952. The evaporation of water from spiders. Journal of Experimental Biology. 29:571-582.

DeVito, J. & D.R. Formanowicz, Jr. 2003. The effects of size, sex, and reproductive condition on thermal and desiccation stress in a riparian spider (Pirata sedentarius, Araneae, Lycosidae). Journal of Arachnology 31:278-284.

Edmonds, D.T. & F. Vollrath, 1992. The contribution of atmospheric water vapour to the formation and efficiency of a spider’s capture web. Proceedings: Biological Sciences. 248:145-148.

Edney, E.B. 1977. Water balance in land arthropods. Springer, New York.

Edwards, R.L., E.H. Edwards & A.D. Edwards. 2003. Observations of Theotima minutissimus (Araneae, Ochyroceratidae), a parthenogenetic spider. Journal of Arachnology 31:274-277.

Ehn, R. & H. Tichy. 1994. Hygro- and thermoreceptive tarsal organ in the spider Cupiennius salei. Journal of Comparative Physiology A. 174:345-350.

Ellis, C.H. 1944. The mechanism of extension in the legs of spiders. Biological Bulletin. Wood’s Hole. 86:41-50.

Fairbanks, L.D. & G.E. Burch. 1970. Rate of water loss and water and fat content of adult Drosophila melanogaster of different ages. Journal of Insect Physiology. 16:1429-1436.

Fielden, L.J., B.R. Krasnov, K.M. Still, & I.S. Khokhlova. 2002. Water balance in two species of desert fleas, Xenopsylla ramesis and X. conformis (Siphonaptera: Pulicidae). Journal of Medical Entomology. 39:875-881.

Foelix, R.F. 1996. Biology of Spiders. Oxford University Press. New York, NY.

Foo, C.W.P., E. Bini, J. Hensman, D.P. Knight, R.V. Lewis, & D.L. Kaplan. Role of pH and charge on silk protein assembly in insects and spiders. Applied Physics A: Materials Science & Processing. Springer Berlin, Heidelberg, Germany.

Gaede, K. & W. Knülle. 1997. On the mechanism of water vapou sorption from unsaturated atmospheres by ticks. The Journal of Experimental Biology. 200:1491-1498.

García-González, F. & L.W. Simmons. 2005. The evolution of polyandry: intrinsic sire effects contribute to embryo viability. Journal of Evolutionary Biology. 18:1097- 1103.

Hadley, N.F. 1994. Water Relations of Terrestrial Arthropods. Academic Press San Diego, CA.

Hadley, N.F., G.A. Ahearn, & F.G. Howarth. 1981. Water metabolic relations of cave- adapted and epigean lycosid spiders in Hawaii. Journal of Arachnology 9:215- 222.

Hebets, E.A. & G.W. Uetz. 2000. Leg ornamentation and the efficacy of courtship display in four species of wolf spider (Araneae: Lycosidae). Behavioral Ecology and Sociobiology. 47:280-286.

Higgins, L.E., M.A. Townley, E.K. Tillinghast & M.A. Rankin. 2001. Variation in the chemical composition of orb webs built by the spider Nephila clavipes (Araneae, Tetragnathidae). The Journal of Arachnology. 29:82-94.

Humphreys, W.F. 1975. The influence of burrowing and thermoregulatory behaviour on the water relations of Geolycosa godeffroyi (Araneae: Lycosidae), an Australian wolf spider. Oecologia 21:291-311.

Kaston, B.J. 1965. Some little known aspects of spider behavior. American Midland Naturalist 73:336-356.

Kaston, B.J. 1981. Spiders of Conneticut. Bulletin 70. State Geological and Natural History Survey of Connecticut. Hartford, CT.

Machin, J., 1976. Passive exchanges during water vapour absorption in mealworms (Tenebrio molitor): a new approach to studying the phenomenon. Journal of Experimental Biology. 65:603-615.

Marshall, S.D. & A.L. Rypstra. 1999. Patterns in the distribution of two wolf spiders (Araneae: Lycosidae) in two soybean agroecosystems. Environmental Entomology. 28:1052-1059.

Marshall, S.D., D.M. Pavuk & A.L. Rypstra. 2002. A comparative study of phenology and daily activity patterns in the wolf spiders Pardosa milvina and Hogna helluo in soybean agroecosystems in southwestern Ohio (Araneae, Lycosidae). The Journal of Arachnology. 30:503-510.

Millot, J. & M. Fontaine. 1937. La teneur en eau des araneides. Bulletin de la Societe Zoologique de France. 62:113-119.

Miner, C.S & N.N. Dalton, eds. 1953. Glycerol. Reinhold. New York.

Montgomery, T.H. 1903. Studies on the habits of spiders, particularly those of the mating period. Proceedings of the Academy of Natural Sciences of Philadelphia. 55:59- 149.

Montgomery, T.H. 1907. On parthenogenesis in spiders. Biological Bulletin 13:302-305.

Needham, G.R. & P.D. Teel. 1986. Water balance by ticks between blood meals. Pp. 100-151. In Morphology, Physiology and Behavioral biology of ticks. (J.R. Sauer & J.A. Hair, eds.). Ellis Horwood Limited, Chichester, England.

Needham, G.R. & P.D. Teel. 1991. Off-host physiological ecology of ixodid ticks. Annual Review of Entomology. 36:659-81.

Needham, G.R. 2009. Personal communication.

Parry, D.A. 1954. On the drinking of soil capillary water by spiders. Journal of Exp. Biology 31:218-227.

Parry, D.A. 1957. Spider leg muscles and the autonomy mechanism. Quarterly Journal of Microscopical Science. 98:331-340.

Phillips, E.F. 1903. A review of parthenogenesis. Proceedings of the American Philosophical Society 42:275-345.

Pulz, R. 1987. Thermal and water relations. Pp. 26-55. In Ecophysiology of Spiders. (W. Nentwig, ed.). Springer-Verlag, New York, New York.

Roland, C. 1984. Chemical signals bound to the silk in spider communication (Arachnida, Araneae). Journal of Arachnology 11:309-314.

Sigal, M.D., J.A. Yoder & G.R. Needham. 1999. Palp-splaying behavior and a specific mouthpart site associated with active water uptake in Amblyomma americanum (Acari:Ixodidae). Journal of Medical Entomology 36:365-369.

Taylor, P.W. 1998. Dragline-mediated mate-searching in Trite planiceps (Araneae, Salticidae). Journal of Arachnology 26:330-334.

Thiemann, T., L.J. Fielden, & M.I. Kelrick. 2003. Water uptake in the cat flea Ctenocephalides felis (Pulicidae: Siphonaptera). Journal of Insect Physiology. 49:1085-1092.

Vollrath, F., Downes, M. & Krackow, S. 1997. Design variability in web geometry of an orb-weaving spider. Physiology and Behavior 62:735-743.

Walker, S.E. & J.T. Irwin. 2006. Sexual dimorphism in the metabolic rate of two species of wolf spider (Araneae, lycosidae). Journal of Arachnology 34:368-373.

Walker, S.E., S.D. Marshall, A.L. Rypstra & D.H. Taylor. 1999. The effects of hunger on locomotory behaviour in two species of wolf spider (Araneae, Lycosidae). Animal Behaviour. 58:515-520.

Wharton, G.W. 1985. Water balance in insects. In: G.A. Kerkut and L.I. Gilbert, eds., Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol.4, pp. 565-601.

Yoder, J.A., M.E. Selim, & G.R. Needham. 1997. Impact of feeding, molting and relative humidity on cuticular wax deposition and water loss in the lone star tick, Amblyomma americanum. Journal of Insect Physiology. 43:547-551.