*Museum of Comparative Zoology, Harvard University;
Department of Chemical Engineering and Biotechnology Center, Tufts University;
European Synchrotron Radiation Facility, Grenoble, France;
§Department of Zoology, University of Melbourne, Victoria, Australia;
and
||Arthur D. Little, Cambridge, Massachusetts
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Abstract |
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Introduction |
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Four hypotheses have been advanced to explain the diversity and evolution of silks spun by the Araneoidea: (1) random genetic events (Rudall and Kenchington 1971
), (2) selection for synthetic efficiency (Mita et al. 1988
; Candelas et al. 1990
; Hayashi and Lewis 1998
), (3) selection for mechanical properties such as strength and elasticity (Denny 1976, 1980
; Gosline, Demont, and Denny 1984
; Craig 1987
; Gosline et al. 1993
; Kohler and Vollrath 1995
); and (4) selection for amino acids that reflect the silk producer's prey (Craig et al. 1999
). None of these hypotheses is exclusive of the others, as each seems to point to a different aspect of silk synthesis or function. For example, the three different types of fibroins that spiders spin are made of many regions of highly repetitive amino acids that make the probability of random deletions or duplications of DNA segments during recombination high (hypothesis 1). Random errors during replication could result in divergent protein compositions and structures affecting positive selection for silks with different structures (hypotheses 2 and 3). Different mechanical properties of silks translate into different mechanical properties of webs that, in turn, bias the prey that webs intercept and, hence, the pool of amino acids available to the spider (hypothesis 4). Spiders, which retain flexible silk-producing systems that allow the composition of silks produced in a single gland to vary, will be better able to optimize the resources available to them.
Greenstone (1979) found that spiders obtain different mixtures of nutrients from different insects and that the hunting spider, Pardosa ramulosa, forages selectively to optimize them. More specifically, Sutcliffe (1963) showed that the hemolymphs of hemitabolous insects (i.e., crickets, bugs, aphids) contain a lower diversity and volume of freely circulating amino acids than the hemolymphs of holometabolous insects (i.e., butterflies, bees, flies, beetles). Barker and Lehner (1972)
found free amino acids in the thoraxes of flown honey bees, Apis mellifera, and, in particular, proline, that the bees use for flight energy. Madsen, Shao, and Vollrath (1999)
showed that starvation directly affects silk mechanical properties. Argiope argentata, like all orb-spinning spiders, consumes its web at the end of a feeding period, and the amino acids that make up the silks are broken down and recycled. If some spiders are able to vary the amino acid content of their silk, reflecting diet, then silk represents a historical record of what resources have been available to the spider.
While we cannot yet directly identify which amino acids are gained directly from prey and the spider's response to them, there are numerous nongenomic molecular mechanisms that could give rise to varying proteins. For example, differential splicing from a single gene could generate families of mRNAs with different sequences, as has been shown among silks produced by the larvae of Bombyx mori (Lepidoptera: Bombycidae; reviewed in Grezelak 1995
). Alternatively, in a process akin to antibody production, spider silks could consist of multiple chains of different proteins (as is the case in some B. mori silks characterized to date), with the relative proportions of the two chains, or even their content, altered through combinatorial association. For example, Mita, Ichimura, and James (1994)
found that mRNA from three silk genes in B. mori recombined to produce four proteins (mechanism not identified). To determine the basis of diverse silk proteins requires information on the sequence of the MA gene or cDNA of A. argentata taken from spiders foraging under different environmental conditions. This work is currently in progress. One published test suggesting that diverse silk proteins are potential effects of diet on silk composition is a comparison of the amino acid contents of silks produced by herbivorous insects and predatory spiders. These results suggest a higher consistency in the amino acid composition of silks produced by herbivores, perhaps due to diet predictability, than in that of the silks produced by predators, perhaps due to unpredictable diets that vary with microhabitat (Craig et al. 1999
).
In this work, we take a first step toward testing the hypothesis that diet affects the amino acid composition of silks by determining (1) if the composition of silks produced in the same glands by conspecific spiders can vary, (2) if differences in spider diet correlate with the amino acid composition of silk, and (3) if variation in amino acid content affects the structure of silk proteins. We started by comparing the amino acid composition of dragline silk produced by A. argentata found foraging in different habitats. Then, using Argiope keyserlingi maintained on four different diets, we determined if silk composition was affected by the total mass of prey or the type of prey that spiders were fed. Finally, we compared the X-ray structures of dragline silks that varied in content and were produced by freely foraging A. argentata collected from different habitats.
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Materials and Methods |
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Analysis of the Amino Acid Contents of Dragline Silks Collected from Freely Foraging Spiders
We tried to account for the possibility that silk composition might vary along a fiber (Work 1984
; Work and Young 1987
) by subsampling a cross-section of the dragline for amino acid analysis. The wrapped silk was viewed under a dissecting microscope, and a portion was cut from the card's open notch, over which the total mass of fibers was centered. Therefore, each silk subsample included multiple, randomly selected portions of the collected thread. Amino acid analyses of the subsampled silks were carried out on a Waters-Pico-tag system. The sample fibers were hydrolyzed in 6 N HCl at 100°C for 24 h, neutralized, and derivatized with phenylisothiocyanate. The total amount of silk analyzed was diluted to 25 x 105 and 11 x 102 pmol of amino acids per sample. The derivatized amino acids were separated and quantified by reverse-phase high performance liquid chromatography.
Controlling for Accuracy of Analysis Techniques
All of the samples were analyzed by the same technician on the same instrument. To test for within-laboratory consistency of our analysis, we compared the composition of a silk sampled from the embiid Antipaluria urichis. Even though these analyses were done a year apart (1997, 1998), we found a <1% difference in content (table 1
). We also point out the consistency among the silks sampled from our controlled feeding experiments. Silk samples were taken from spiders fed the same type of food at 14-day intervals. The average differences in serine, alanine, and glycine before and after treatments were 0.14, -0.08, and -0.03 percentage points, respectively.
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Analysis of the Composition of Silks Produced by Spiders Maintained on Diets that Differed in Either Mass or Content
Twenty-nine juvenile female A. keyserlingi were collected from multiple sites around Brisbane, Australia, and reared on constant and equal diets of blowflies (Lucillia cuprinai). After reaching sexual maturity, the spiders were transferred to 350-ml plastic cups to prohibit them from spinning a web, and randomly divided into two groups. Group 1 was maintained on the high-prey diet (14 blowflies over 14 days) and then switched to a low-prey diet (5 blowflies over 14 days); group 2 was maintained on the low-prey diet and then switched to the high-prey diet. The spiders were given water daily. After each 14-day treatment, their dragline silks were collected by hand. Only spiders that survived the entire experimental period and that ate all of the flies they were given contributed silks to the final analysis. Amino acid analyses of the silks' contents were carried out as described above.
An additional 22 juvenile females were collected, reared on a constant diet of blowflies, and transferred to 350-ml plastic cups at sexual maturity. The spiders were then randomly allocated to three groups and fed crickets (Acheta domestica), bees (A. mellifera), or blowflies. During the 17-day feeding treatment, the spiders received one meal every three days (a total of five meals, weighing approximately 0.08 g each). We weighed each meal before it was ingested, as well as any prey remains collected the following day. The amount of food ingested by the spiders did not differ among the treatments (fly group: 0.391 ± 0.006 g; cricket group: 0.404 ± 0.006 g; bee group: 0.389 ±0.006 g; F = 1.9, P = 0.17). Spiders were misted daily, and silk samples (dragline silk) were collected by hand using the approach outlined above before and after the 17-day treatments.
Structure of Silk Collected from Freely Foraging Spiders
X-ray diffraction analyses of bundles of silk threads have shown that spider silk is composed of both crystalline and amorphous fractions (Warwicker 1960
; Lucas, Shaw, and Smith 1965
; Grubb and Jelinski 1997
; Riekel et al. 1999
), but the details of the structures have been unresolved. To better resolve the proteins' organization, we used synchrotron radiation X-ray microdiffraction on single silk threads of disparate amino acid compositions (Bram et al. 1997
; Riekel et al. 1997, 1999
).
The X-ray diffraction experiments were performed at beamline ID13 of the European Synchrotron Radiation Facility with a 10 µm beam at a wavelength of 0.0782 nm. Single fibers were optically selected and glued without applied tension to a steel washer, which was mounted on a goniometer head and transferred into the X-ray beam by a three-axis gantry. X-ray diffraction patterns were acquired at 25.5°C with a CCD detector. Ten diffraction patterns from 60 exposures each were recorded along each fiber axis, with a distance of 10 µm between individual points in order to limit radiation damage. The detector-to-sample distance was calibrated using an Al2O3 standard and was found to be 56.21 mm.
The unit cell parameters of the X-ray images were determined by fitting each pattern with the XFIX program from the CCP13 package (http://www.dl.ac.uk/SRS/CCP13; Daresbury Laboratory, 1997). Radial intensity profiles were simulated by one-dimensional Gaussian functions using the FIT2D package (http://www.esrf.fr/computing/expg). Particle size values were calculated by the Scherrer formula, taking the instrumental broadening into consideration. For a simulation of the azimuthal extent of reflections and amorphous halo, two-dimensional Gaussian functions were used. The simulation for the "Trinidad" species was based on three Gaussian functions corresponding to the (020)/(210) Bragg reflections and the amorphous halo. Convergence was not obtained for the "Anguilla" species due to limited counting statistics. The orientation distribution along the fiber axis, fc, was calculated from Herman's orientation function (fc = 0.5 x 3cos2
1
- 1) based on the equatorial (020)/(120) reflections (Bram et al. 1997
).
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Results |
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There were no differences in the amount of weight the spiders gained during the feeding treatment (fly group: 0.074 ± 0.001 g; cricket group: 0.089 ± 0.009 g; bees: 0.075 ± 0.009 g; F = 1.03, P = 0.38). When spiders were switched from a diet of flies to a diet of bees, the amino acid content of their silks did not change. When spiders were switched from flies to crickets, however, the serine content of their silks increased. While the sample size was too small to accurately compare the percentage increase of the amino acids using parametric procedures, a nonparametric test was used to determine if the location of the distribution of the differences differed. A matched-pair, signed-rank test (Mann-Whitney, U = 0, Npairs = 12, P < 0.025; table 2 ) showed that spiders fed crickets produced silks with significantly higher serine contents.
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Discussion |
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In an effort to control the rate at which the silk is spun, some investigators have employed forcible silking, in which spiders are anesthetized and their legs are taped to a substrate to restrain them. Anesthetization, however, affects both the neural control of the gland and the intragland environment. Furthermore, the silks are reeled mechanically at a rate that is set by the investigator. The forced-silking approach completely eliminates the spider's spinning behavior. Due to these artificial conditions, the silks that are collected may not be "normal," as investigators from several laboratories have shown (Work 1977
; Magoshi, Magoshi, and Nakamura 1994
; Madsen and Vollrath 2000
). Using X-ray microdiffraction, one of us (Riekel, Mueller, and Vollrath 1999
) has studied the effects of forced silking in detail.
Because we were interested not only in the amino acid contents of silks, but also in the structural organization of the protein threads, we chose to sample silks from freely spinning spiders ("natural silking"). Masden and Vollrath (2000)
measured the effects of anesthetization on dragline silks produced by Nephila during forced silking. They showed that, in comparison to control silk, silks produced by spiders anesthetized with CO2 had a significantly lower breaking strain and breaking energy and a significantly higher initial modulus. At the onset of anesthesia, silk diameter became highly variable or fibrillated and fibers produced by higher reeling speeds resulted in increased breaking stress and decreased breaking elongation (Madsen, Shao, and Vollrath 1999
). Because we allowed the spider to maintain a normally functioning neural and internal environment and to control the rate at which it spun the silk, we believe that the structure and composition of our silk samples are likely to be more representative of the characteristics of natural silks than those that can be obtained via forced silking.
Some investigators have noted that dragline silk (from the ampullate gland) spun by Nephila under forced-silking conditions can become contaminated with silk from adjacent glands. In particular, with the aid of a microscope, Work and Young (1987)
observed that when the spigot of the ampullate gland is stimulated to produce dragline silk, any slight brush against an adjacent spigot (leading to other glands) will stimulate them to secrete proteins as well. As a result, dragline silks can become contaminated with other silk proteins. While it is not possible to see the spigots from which silks are secreted when silks are collected naturally, the observations of Work and Young (1987)
suggest that contamination of threads is the result of inadvertent manipulation stimulating the incorrect spigot and not a behavior one would expect of a spider in a nonstressed situation. While natural silking does not allow silk-secreting spigots to be viewed when the fibers are collected, it is possible to minimize the possibility of contamination by observing the reflectance properties of the silk thread, its material properties, and the number of silk threads the spider releases. In particular, aciniform gland silks produced by Argiope species are white, highly reflective, and secreted in a ribbon composed of multiple silk strands. Furthermore, when aciniform silk is "dry spun" or secreted into the air, it pulls apart, as would a ball of unspun cotton. Dragline silks, however, are secreted appearing as a single thread even though they are actually made up to two fibers. Even dry spun, the silk can support the spider's weight. The silks are translucent and difficult to see unless viewed from an appropriate angle or against a dark background and can support the spider's weight. When collecting silks from Argiope, we observed only one case in which aciniform gland silks were released; this was in response to annoying pokes with forceps by the experimenter, and the sample was not included in the analysis.
Because of our need to make sure that the silks we collected were not contaminated, we compared the amino acid composition of dragline silk (ampullate gland) and decoration silk (from aciniform gland) produced by two different species of Argiope, A. appensa and A. trifasciata. Both dragline silks were collected by the same investigator using the natural-silking technique on two different occasions. The aciniform gland silks were sampled directly from the spiders' webs (table 1 ). The data show that the composition of aciniform silks is very different from that of dragline silks. Aciniform silks contain approximately equal amounts of alanine, glycine, and serine, while ampullate gland silks contain roughly 300% more alanine and glycine. While we cannot conclusively state that none of the silks we collected were contaminated, the range of variation in MA silk content is well beyond the compositions of aciniform silks that we measured. Furthermore, we point out that in our high-prey and low-prey diet experiments, we showed that fractional differences in serine, alanine, and glycine all differed by much less than 1% (table 2 ). We believe that the need to collect proteins that accurately represent the structural properties of the silk, along with our observations that showed no significant change in silk content when spiders were maintained on diets invariant in composition and silked 2 weeks apart, suggests that natural silking does not result in fiber contamination but does yield fibers of realistic mechanical properties.
Proteins that are highly expressed and are composed of amino acids, which are costly to synthesize, are likely to place a greater drain on an animal's energy resources than proteins composed of amino acids that are metabolically simple to produce or ingested directly from prey (Craig et al. 1999
). In the absence of constraint, natural selection should favor the evolution of silk proteins that are relatively "inexpensive" to produce. We have taken a first step toward testing this idea by comparing the composition of dragline silks produced by A. argentata foraging in different types of vegetation throughout the Caribbean. We found that silks varied inversely in the amount glycine and serine they contained. We then completed a series of experiments in which A. keyserlingi was maintained under different dietary regimes and compared the amino acid compositions of their silk before and after treatment. Our data show that the compositions of dragline silks spun by A. keyserlingi are not affected by the volume of the spider's diet, but do suggest that, at least in a preliminary fashion, silk composition is affected by the type of prey on which the spider is fed. When we compared the structures of silks spun by A. argentata via X-ray diffraction, we found that the changes in their amino acid compositions did not affect the crystalline domain of the protein, the region that determines silk strength, and therefore must have been localized to the amorphous domain of the protein, the region that determines silk elasticity. Hence, even though we have not yet untangled the balance of selective effects on silk function (hypothesis 3) from the selective effects on silk composition (hypothesis 4), our data do suggest that the differences we observed among the dragline silks collected from free-foraging spiders are diet-related.
Studies on the nutritional ecology of arthropods, popular in the 1960s and 1970s, generally compared the foraging patterns of insects to the distribution resources they used and the nutrients they obtained. One example of this type of study resulted in the discovery of what is now accepted as the strong evolutionary relationship between plant type and pollinator behavior (Baker and Baker 1966
). Plants secrete different concentrations of sugars (nectars), amino acids (nectar and pollen), and fats (nectar and oils) through which they attract a narrow or broad range of pollinators (i.e., 35, 36). For example, flowers pollinated by hummingbirds (which feed additionally on insects for protein) have high concentrations of sugars but low concentrations of amino acids. Flowers that attract butterflies, however, have higher amino acid contents (Inouye and Waller 1984
). Even closely related plants may display large differences in nectar secretions (Schemske 1980
) or amino acid compositions (Rust 1977
).
One approach to addressing the relationship between diet and silk content is to compare the amino acid compositions of silks produced by herbivores and carnivores. The most prolific silk-spinning arthropods next to spiders are the herbivorous moth (Lepidoptera) larvae that spin cocoons. These larvae, such as B. mori, have evolved to produce silks in a simple one-gland system that occupies 60% of their body mass just prior to pupation (Prudhomme et al. 1985
). The source of larval food is selected by the mother when she lays her eggs on a specific food plant. After hatching, the larvae can spend their entire preadult period, and, hence, their silk-producing life span, on the same plant. While the amino acid diversity of their diets is low, the caterpillars are assured of a predictable and unlimited supply of carbohydrates. Diet constancy is reflected in the relatively invariant composition of silk produced by herbivores (Craig et al. 1999
). In comparison to the diet of an herbivore, the diet of a predator is unpredictable, diverse in amino acids but carbohydrate-poor. This diet variability may be reflected in the multiple types of silks a spider produces, as well as variation in the composition of silks drawn from the same gland. Because newly hatched spiderlings, and in particular those in the genus Argiope, disperse broadly before spinning even their first web, the ability to vary silk composition would allow them to adapt more effectively to new habitats.
We do not know the genetic limitations on variability of silk proteins or the mechanism by which variable silks are produced. Manning and Gage (1980)
, however, found 22 different alleles for silks produced by inbred stocks of B. mori that they attributed to unequal crossover. To date, investigators of the molecular genetics of silk proteins have not considered silks collected from the same species of spider found foraging in different ecological contexts. Recent research on the molecular genetics of ecological diversification, however, suggests that other organisms adapt secreted proteins to available prey. For example, Duda and Palumbi (1999)
identified diverse and multiple conotoxins produced by Conus abbreviatus and found that the toxins exhibited a higher rate of evolution than most other known proteins. They suggested that toxin diversity was the product of gene duplication and diversifying selection and proposed that diverse toxins may have evolved, allowing gastropods to target different types of prey characterized by diverse ion channels and neuronal receptors.
Molecular genetic analyses of both dragline (Xu and Lewis 1990
) and flagelliform (Hayashi and Lewis 1998
) silks, the primary proteins that make up orb webs, have led investigators to conclude that the characteristic, highly repetitive sequences that make up silk proteins evolved via unequal cross-over. Analogous to toxin proteins, it could be that the within-species diversity of dragline silks is the product of positive, diversifying selection for silks with specific functional properties. Alternatively, posttranscriptional editing of expressed sequences would be a way of attaining compositional diversity while allowing spiders to retain a single silk gene template. Because spider orb webs capture prey by intercepting and absorbing their kinetic energy during flight (i.e., Denny 1976
; Craig 1987
; Vollrath and Edmonds 1989
), differences in silk structure (and, hence, the energy absorbing properties of silks) may be a primary mechanism of trophic diversification among web-spinners.
Over the past 10 years, there has been an intense interest in silk among investigators hoping to manufacture artificial polypeptides by making use of spider silk sequence and design. At present, we are limited by molecular data largely drawn from N. clavipes (Xu and Lewis 1990
; Hinman and Lewis 1992
; Lewis et al. 1996
; Hayashi and Lewis 1998, 2000
) and Araneus diadematus (Guerett et al. 1996
). Unfortunately, there are no additional data on silks collected from spiders foraging in different environments, particularly from N. clavipes, even though they are found in almost as many sites and in different ecological environments as Argiope argentata. Nevertheless, if we do find that individual spiders of any taxa are able to modify the composition and functional properties of silks produced in the same gland, this would suggest alternative avenues through which to study biosynthetic silk synthesis and, perhaps, lead to a new understanding of a process variation that has significantly affected the evolution of the Araneoidea.
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Acknowledgements |
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Footnotes |
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1 Keywords: spider
silk
Argiope argentata,
amino acid
protein structure
diet
2 Address for correspondence and reprints: Catherine L. Craig, Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge, Massachusetts 02138. E-mail: ccraig{at}oeb.harvard.edu
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