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First published online March 30, 2006
Journal of Experimental Biology 209, 1463-1486 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02147

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Changes in composition of spider orb web sticky droplets with starvation and web removal, and synthesis of sticky droplet compounds

Mark A. Townley^1,*, Edward K. Tillinghast¹ and Christopher D. Neefus²

¹ Department of Zoology, University of New Hampshire, 46 College Road, Durham, NH 03824-2617, USA
² Department of Plant Biology, University of New Hampshire, 46 College Road, Durham, NH 03824-2617, USA

^* Author for correspondence (e-mail: mtownley{at}cisunix.unh.edu)

Accepted 6 January 2006

	Summary

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
The sticky spiral of araneoid spider orb webs consists of silk fibers coated with adhesive droplets. The droplets contain a variety of low-molecular-mass compounds (LMM). Within a species, a fairly consistent ratio of LMM is often observed, but substantial variability can exist. To gain insight into factors influencing LMM composition, spiders of three araneid species were starved and LMM from their webs were analyzed for changes in composition. To determine if these changes were consistent with the spider's ability to synthesize the different organic LMM, synthetic capacities were estimated following the feeding of radiolabeled metabolites. Some changes in droplet composition were broadly consistent with differing synthetic capacities: molar percentages of less readily synthesized compounds (e.g. choline, isethionate, N-acetyltaurine) typically declined with starvation, at least during a portion of the imposed fast, while more readily synthesized compounds (e.g. GABamide, glycine) tended to increase. Most striking was the apparent partial substitution of N-acetylputrescine by the more readily synthesized GABamide in fasting Argiope trifasciata. However, departures from expected compositional shifts demonstrated that synthetic capacity alone does not adequately predict sticky droplet compositional shifts with starvation. Moreover, feeding controls exhibited some changes in composition similar to starving spiders. As the webs of both feeding and starving spiders were removed for chemical analysis and could not be recycled, the loss of LMM contained in these webs likely contributed to similarities between treatments. In addition, feeding spiders molted, oviposited and/or built heavier webs. The added metabolic demands of these activities may have contributed to changes in composition similar to those resulting from starvation.

Key words: 4-aminobutyramide, Araneus cavaticus, choline, glycine, glycine betaine, isethionic acid, orb web, putrescine, resource allocation, spider web recycling, sticky spiral, taurine

	Introduction

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
In the construction of its orb web an araneoid spider first builds a non-sticky scaffolding using ampullate silk gland fibers and piriform silk gland junctional cements/attachment disks. One part of the scaffolding, the radii, are arranged like the spokes of a wheel. Subsequently, the sticky spiral (or adhesive or viscid spiral, among other names) (Zschokke, 1999) is attached to the radii. The sticky spiral consists of a pair of flagelliform silk gland fibers coated with an adhesive, aqueous secretion from the aggregate glands (Sekiguchi, 1952; Peters, 1955). This aggregate gland secretion contains organic and inorganic low-molecular-mass compounds (LMM; <200 Da), at least one high-molecular-mass phosphorylated glycoprotein and, likely, lipids (see references in Higgins et al., 2001; Schulz, 2001). Organic LMM include 11 of the 12 compounds monitored in this study; GABamide (4-aminobutyramide; GAB), N-monoacetylputrescine (NAP), isethionic acid (2-hydroxyethane sulfonic acid; Ise), N-acetyltaurine (NAT), glycine (Gly), choline (Cho), glycine betaine (Bet), alanine (Ala), proline (Pro), putrescine (Put), taurine (Tau) (Fischer and Brander, 1960; Tillinghast and Christenson, 1984; Vollrath et al., 1990; Townley et al., 1991; Higgins et al., 2001).

The aggregate gland secretion, initially deposited as an unstable liquid cylinder, spontaneously redistributes into a pattern of linked droplets (Boys, 1889; Warburton, 1890; Edmonds and Vollrath, 1992; Ball, 1999). Concurrent with droplet formation is the formation of discrete nodules at the center of the droplets that contain the aforementioned glycoprotein (Vollrath and Tillinghast, 1991). While it is believed the nodules are the principal adhesive agents in the droplets (Richter, 1956; Vollrath and Tillinghast, 1991), the functions of the LMM have not been established, though one role, adsorbing atmospheric moisture, is indicated by the hygroscopic properties of certain LMM (Fischer and Brander, 1960; Schildknecht et al., 1972; Vollrath et al., 1990; Townley et al., 1991).

The LMM account for about 40–70% of the desiccated mass of orb webs, indicating they are important to web function (Fischer and Brander, 1960; Anderson and Tillinghast, 1980; Tillinghast, 1984; Tillinghast and Christenson, 1984; Townley et al., 1991). They may contribute to the elastomeric mechanical properties of the sticky spiral; directly, by inhibiting crystallization within the flagelliform fibers (Gosline et al., 1995), or indirectly, by adsorbing moisture, thereby keeping the fibers elastomeric through hydration (Vollrath and Edmonds, 1989; Bonthrone et al., 1992; Hayashi and Lewis, 1998). Direct and indirect effects of the LMM may also be important to the functioning of the adhesive glycoprotein within the nodules. The conformation adopted by the glycoprotein and the formation of nodules may depend on direct interactions between the glycoprotein and LMM, and the ability of the glycoprotein to spread out on contact with an insect (Richter, 1956; Vollrath and Tillinghast, 1991) presumably relies on the presence of water. Water adsorbed by LMM also promotes droplet formation, which increases adhesiveness (Edmonds and Vollrath, 1992), and contributes to aerodynamic drag following the impact of an insect, thus helping to dissipate the prey's kinetic energy (Lin et al., 1995).

The LMM composition of sticky droplets often differs quantitatively and qualitatively among araneoid species (Vollrath et al., 1990; Townley et al., 1991; Higgins et al., 2001) (M. A. Townley and E. K. Tillinghast, unpublished). The significance of these differences is unknown. One attempt to determine if differences in composition translate into differences in web hygroscopicity among three araneid species did not demonstrate such a relationship (Townley et al., 1991). Nor do we know the extent to which the composition of the droplets is tailored to the physical environment in which a spider forages, the prey captured, or the metabolic needs of the spider. And while LMM composition within a species is consistent enough that analyses of pooled web collections from groups of individuals generally yield similar results (Vollrath et al., 1990; Townley et al., 1991), substantial intraspecific differences have also been observed within and among populations, between the sexes, and following a change in environment/diet (Vollrath et al., 1990; Townley et al., 1991; Higgins et al., 2001).

The goal of the present study was to examine the influence of starvation on the LMM composition of the sticky droplets and to determine if observed changes reflect differences in the spider's capacity to synthesize the various organic LMM. Specifically, we anticipated that the total mass of LMM would decrease in webs of starving spiders, but that there would be greater relative declines in those organic LMM the spider is less able to synthesize. Therefore, in addition to analyzing series of webs built by starving and feeding spiders of three araneid species, we fed radiolabeled compounds to two of these species to determine to what extent the spiders can synthesize the different organic LMM (Kasting and McGinnis, 1966). Given the results of these synthetic capacity measurements, some of the changes in composition observed with starvation conformed to our expectations, but others did not. Unexpected results also came from the control feeding spiders, which also exhibited changes in droplet composition, in some respects similar to trends seen in webs of starving spiders. Possible explanations for these results are discussed. In addition, the construction of egg sacs by some of the study spiders allowed us to make a preliminary examination of the influence of the egg laying cycle on droplet composition.

	Materials and methods

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
Synthesis of organic LMM by Argiope
Spider collection, maintenance and radioisotope feeding
Adult female Argiope aurantia Lucas 1833 and Argiope trifasciata (Forskål 1775) were collected in southern New Hampshire, USA from late July to late August and in the latter half of September, respectively. Spiders were housed individually in wood or aluminum frames (51 cmx51 cmx9 cm) with glass plates on the front and back. They were exposed to the prevailing natural light:dark cycle in a room facing east at temperatures and relative humidities that approximated outside conditions.

Four groups of A. aurantia (5–7 spiders/group) and three groups of A. trifasciata (6–7 spiders/group) were formed. For each A. aurantia, 1.1x10⁵ Bq D-[U-¹⁴C]glucose (ICN Biomedicals, Irvine, CA, USA) in 3 µl water was placed on the mouthparts using a 10 µl Hamilton syringe (Reno, NV, USA) and the spider was observed until the droplet was completely imbibed. Argiope trifasciata were likewise fed D-[U-¹⁴C]glucose, but in quantities of 3.7x10⁴–1.1x10⁵ Bq/spider.

The synthesis of the three sulfonic acids in the webs' sticky droplets (Ise, NAT, Tau) was also investigated by feeding adult female A. aurantia (4 groups; 4–6 spiders/group; 3.7x10⁴–1.5x10⁵ Bq/spider) a solution containing L-[³⁵S]methionine and L-[³⁵S]cysteine (TRAN³⁵S-Label, ICN).

Spiders were either fed radioisotope within 3 days of being captured or, if held longer prior to isotope feeding, were fed crickets or grasshoppers. They were not fed after isotope feeding, but orbs built on the day of isotope feeding were partially collapsed and the spiders were allowed to recycle them. Water was given daily, except for the day before and the day of isotope feeding to encourage drinking of the labeled solution.

Handling of radiolabeled orb webs
The first five webs built by each spider after ingesting radioisotope were collapsed, wound onto one end of a glass micropipet, and stored at –20°C. All webs built by members of the same group were pooled on a single micropipet (19–30 webs/group). Not all spiders built five webs. While still on their micropipets, each group's web collection was extracted twice in 4 ml distilled water for 1.5 h with occasional gentle vortexing. The two extracts were pooled, dried and analyzed by proton nuclear magnetic resonance spectroscopy (¹H-NMR) as described below using the Argiope acquisition parameters (see `¹H-NMR analysis'). From these analyses molar percentages of the 11 organic LMM (listed in the Introduction) in each of the 11 extracts (seven ¹⁴C-labeled, four ³⁵S-labeled) were calculated. Extracts were then fractionated individually by high-voltage paper electrophoresis (HVE).

HVE and chromatography of radiolabeled water-soluble web fractions
Each water-soluble extract was fractionated by HVE at 3000 V on 23 cmx57 cm sheets of Whatman 3MM chromatography paper (Brentford, England), with the extract applied in 75 µl of electrolyte solution over a 13 cm long origin. For the ¹⁴C-labeled extracts, the origin was 28 cm from the positive pole and electrophoresis was carried out for 35 min. For the ³⁵S-labeled extracts, the origin was 41 cm from the positive pole and the run lasted 55 min. The electrolyte solution used was pyridine:glacial acetic acid:water (133:4.6:1862.4, v/v), pH 6.4. Coolant at 2°C was passed through the unit's lower plate.

After electrophoresis, the paper was air dried and autoradiograms were generated using Kodak BioMax MR film (Rochester, NY, USA). Using the autoradiograms as a guide, the electrophoretograms were cut into radioactive and nonradioactive bands. All bands were eluted overnight with distilled water containing 0.001% sodium azide in a chamber saturated with water vapor. Eluates were dried, resuspended in 1 ml distilled water, and 50 µl removed for scintillation counting. Selected eluates were examined by ¹H-NMR to establish locations of organic LMM.

To determine what percentage of the radioisotope in a HVE eluate was incorporated into an organic LMM of interest, portions of radioactive eluates were further fractionated by two-dimensional thin layer chromatography (2D-TLC) on 20 cmx20 cm cellulose plates (0.1 mm thickness, Merck KGaA, Darmstadt, Germany) using pyridine:acetone:ammonium hydroxide:water (45:30:5:20, v/v) in the first dimension and 2-propanol:formic acid:water (75:12.5:12.5, v/v) in the second dimension (Schmidt, 1974). This was necessary because HVE did not resolve all of the organic LMM measured in this study and because these compounds, though they constitute the bulk of the organic LMM, could not be assumed to be the only organic or sulfur-containing compounds extracted by water from orb webs (and, indeed, they are not). Autoradiograms were prepared from the 2D-TLC plates as above. If an autoradiogram revealed two or more radioactive compounds, these were individually scraped off the plate and their radioactivities measured by scintillation counting. Identifications of the organic LMM on the plates were based on NMR of the HVE eluates and on experience in this system with the migration characteristics of most of the organic LMM of interest. Where any doubt remained, radioactive compounds were scraped off plates and examined by NMR to confirm identity.

Using pre- and post-fractionation NMR data and the radioactivity data obtained following HVE and 2D-TLC, relative specific radioactivities for the organic LMM were estimated (initially in c.p.m./mole%, then, for ¹⁴C-labeled LMM, converted to c.p.m./molar quantity of carbon).

For the two N-acetylated LMM, NAP and NAT, estimates were also made of the relative specific activities of their acetyl groups versus their Put or Tau moieties. Because NAP was not detected in webs of A. aurantia, only the three A. trifasciata water-soluble extracts were used to make this determination for this compound. After identifying the HVE eluates containing NAP and NAT by NMR, 70% of each was hydrolyzed under vacuum (6.7 Pa) in 6 mol l^–1 HCl at 115°C for 20 h. At the same time, commercial samples of Put and Tau (two each) were treated likewise. Based on the percentage recovery of these standards (Put, 83.8%; Tau, 84.3%), a correction for losses occurring during hydrolysis was made. Hydrolyzed and unhydrolyzed portions of eluates were fractionated by HVE and 2D-TLC and autoradiograms generated. Of the total ¹⁴C present in these eluates, the percentage incorporated into the Put/Tau moieties versus the intact compounds was determined by scintillation counting of compounds scraped off the 2D-TLC plates. The difference between these was taken to be the percentage of the ¹⁴C incorporated into the acetyl groups.

Effects of starvation and web removal on orb web mass and LMM composition in Araneus and Argiope
Collection and maintenance of spiders
Juvenile Araneus cavaticus (Keyserling 1882) were collected from barns in southern New Hampshire, USA between early April and mid-July. Juvenile female Argiope aurantia and Argiope trifasciata were also collected locally from mid-July to early August and throughout August, respectively. Spiders were housed as described above.

Before spiders molted and were placed in an experimental group, they were fed 1–3 flies on days they built webs, were allowed to recycle (i.e. consume) their webs freely, and were given water daily. Both before and after assigning spiders to groups, A. cavaticus and A. aurantia were fed house flies (Musca domestica L.) and other dipterans [primarily Phaenicia sericata (Meigen 1826) and Phormia regina (Meigen 1826)], while A. trifasciata received house flies exclusively.

Formation of study groups and collection of orb webs
During this study, 21 groups were formed (supplementary material Fig. S1), with the spiders composing a group receiving the same treatment (fed or starved) and being of the same species, sex and stage (juvenile or adult) and, for adult females, having made the same number of egg sacs (0–3). All spiders were added to a group at the same point in the molt/intermolt cycle (beginning of intermolt). With A. cavaticus, webs of male and female juveniles (all penultimate instars) and adult females were collected. With the two Argiope species, only webs built by adult females were collected. An individual spider could belong to only one group at a time, but to two or more groups over the course of the study, as explained below.

Orb webs were collected from a spider only once it had reached the desired stage following its most recent ecdysis. From 1–3 molts were required in the laboratory before the desired stage was reached. Spiders were initially divided into a feeding group and a starving group, with the first individual to reach the desired stage randomly assigned to one of these groups. The next individual that molted to the desired stage was then assigned to the other group and this alternation continued as other spiders subsequently molted to the desired stage.

Following ecdysis, with the building of the first orb web, the spider, whether assigned to a feeding or starving group, was fed two or more flies (see above) totaling about 50 mg wet mass. The web was then partially collapsed and the spider was allowed to recycle it. Thus, no compositional or mass data were obtained from the first web. All subsequent webs, however, were collected on micropipets and stored at –20°C. All second webs built by members of the same group were collected on the same micropipet and are referred to as the web 2 collection of the group. Likewise, all third webs built by the spiders within a group were pooled to yield the web 3 collection and so on. The complete set of web collections from a group is referred to as a `series'.

Spiders in starving groups were treated differently from spiders in feeding groups after the construction of the second orb web. In both groups, the second webs were collected as described above, but then only feeding group spiders were handed one or more flies, totaling about 25–35 mg wet mass. Feeding group spiders were fed only on days they built a web. All spiders were given water daily. With starving groups, spiders were stressed until resultant changes in behavior/physiology were readily apparent (e.g. sluggishness, construction of incomplete webs), then feeding was resumed, at which point the spiders became members of a `resumed feeding' group (supplementary material Fig. S1).

All constructed orb webs, unless they contained no sticky spiral whatsoever, were collected from each member of a group and added to that group's series of web collections until one of four events occurred (supplementary material Fig. S1): (1) the spider was transferred to another group, (2) the spider died, (3) the spider escaped, or (4) the date arrived, 30 September, on which other obligations forced us to end web collecting for the year. This lattermost event was not too detrimental as many of the spiders involved, all adult A. cavaticus or A. aurantia, had already become relatively inactive by this time and would have built few additional webs. However, the series from the A. aurantia PES3 group (see next paragraph) was ended prematurely. Argiope trifasciata webs were collected during a different year when there was no need to terminate the study on a particular date.

There were three reasons for transferring a spider to another group. Transfer of starving group spiders to a resumed feeding group has already been described. The other two reasons were egg sac construction and molting. Egg sac construction resulted in a spider being transferred to a post-egg-sac (PES) group. Because some individuals of both A. aurantia and A. trifasciata produced three egg sacs (containing nonviable eggs), the construction of each of which was followed by the spinning of additional orb webs, there are three successive PES groups for both species (PES1, 2 and 3; supplementary material Fig. S1). Molting pertains to the penultimate instar A. cavaticus only since adults do not molt. On reaching adulthood males lack the ability to build orb webs (Sekiguchi, 1955) so the final molt marked the end of their period of service. Female A. cavaticus, on the other hand, by the procedure described above, were assigned to either a feeding adult or a starving adult group after their final molt (supplementary material Fig. S1).

Following the transfer of a spider to either a resumed feeding or PES group we did not allow it to recycle its first web, as spiders in starving or feeding groups were allowed, but collected this web and fed the spider one or more flies totaling about 25–35 mg wet mass. In all other ways web collection and feeding were carried out as for members of feeding groups.

Gravimetric measurements of orb webs
Each web collection was scraped off its micropipet with a razor blade, desiccated in vacuo over phosphorus pentoxide for at least 2 days, weighed to the nearest 0.01 mg, then extracted three times in 2 ml distilled water for 1 h with occasional gentle vortexing. The three extracts were pooled and dried on a Savant Speed Vac Concentrator (Hicksville, NY, USA), then transferred with two volumes of distilled water totaling 300 µl to a pre-weighed cup fashioned from the cap of a microcentrifuge tube. After desiccating as above for at least 3 days, this water-soluble fraction of the web, containing the LMM, was weighed and then transferred back to its sample tube with three volumes of distilled water totaling 1 ml. After drying, the sample was analyzed by ¹H-NMR. The desiccated water-insoluble fraction was also weighed.

Within a group, not all individuals built the same number of webs (supplementary material Table S1). Consequently, later web collections in a series contained fewer webs than earlier web collections. Initially, therefore, anticipating difficulty with ¹H-NMR analysis on the smaller samples (due to unacceptably large numbers of scans being required to achieve a good signal-to-noise ratio), we typically pooled two or more end-of-series web collections to generate water-soluble extracts that would be large enough to allow for analysis in a reasonable amount of time. Note that this more infrequent `horizontal pooling' of, e.g. web collections 16–19, is in addition to the invariable practice of `vertical pooling' of, e.g. all second webs built by the members of a group. We found, however, that our first horizontal poolings were too generous and that smaller samples (0.3–1 mg) could be analyzed within an acceptable amount of time (3 h to overnight). Thus, we did less horizontal pooling as the study progressed. As the first webs analyzed were those built by Araneus cavaticus, more horizontal pooling was carried out on webs of this species.

Gravimetric measurements of spiders
Fresh and dry mass data on the three study species were obtained to gain some measure of the percentage of a spider's dry mass that it typically invests in its orb web, thus providing an indication of the loss incurred when, as in this study, a spider is denied the opportunity to recycle its web. Freshly captured local penultimate instar and adult female Araneus cavaticus, and adult female Argiope aurantia and Argiope trifasciata were weighed immediately on return to the laboratory. Some of these individuals were anesthetized with CO₂, immersed in 80% ethanol for 1 h, and dried to constant mass in a 43°C oven. Only adult females that were not conspicuously gravid were collected for these measurements.

Composition of orb web water-soluble fractions
¹H-NMR analysis. The water-soluble fraction of each web collection was dissolved in 0.5–1.0 ml 99.96% D₂O (Cambridge Isotope Laboratories, Andover, MA, USA) and analyzed on a Bruker AM-360 spectrometer (Billerica, MA, USA) with a 5 mm proton selective probe. 360-MHz ¹H spectra were obtained at a temperature of 300K with 2-methyl-2-propanol added as an internal standard (=1.2200 p.p.m.). Following Fourier transformation, peak areas were integrated and used to calculate the molar percentages of the 11 organic LMM dealt with in this study. ¹H-NMR chemical shifts and coupling constants for each of these LMM in D₂O have been reported previously (Townley et al., 1991; Higgins et al., 2001).

In all NMR analyses a spectral width of 5000 Hz was examined and a pulse width of 4.3 µs, yielding about a 53° flip angle, was used. Various numbers of scans (128–5064) were accumulated depending on sample size. Several other acquisition parameters differed between A. cavaticus and Argiope samples. With the A. cavaticus extracts, analyzed first, the pulse repetition time was the same as the acquisition time, 3.28 s, during which 32K data points were acquired and later zero-filled to 64K prior to Fourier transformation. However, analyses of a standard solution containing the 11 organic LMM indicated that a longer time between pulses would yield more accurate molar percentages (supplementary material Table S2), particularly for Pro. Therefore, with the Argiope extracts, a longer pulse repetition time was adopted (8.28 s) that still allowed samples to be analyzed within a reasonable amount of instrument time, with 64K data points acquired over 6.55 s, later zero-filled to 128K. We did not attempt to apply corrections to the A. cavaticus data. Thus, more than anything else, Pro is likely somewhat underrepresented in the A. cavaticus webs, though consistently so.

In addition to measuring relative quantities of the organic LMM in molar percentage, we wanted to estimate absolute quantities of the organic LMM. With the A. cavaticus web collections, this was achieved by a standard addition method whereby each sample was analyzed by NMR twice, before and after the addition of synthetic GAB (Kleemann et al., 1980) (1 µmol mg^–1 water-soluble fraction). Integrations from the two analyses allowed us to calculate average µg web^–1 for each organic LMM in each web collection. We also calculated the percentage of the water-soluble fraction's mass that could be accounted for by the 11 organic LMM. For the two Argiope species, we obtained approximate µg web^–1 of the organic LMM by assuming that the percentage of the water-soluble fraction's mass that could be accounted for by the 11 organic LMM was the same as the mean in A. cavaticus.

Phosphorus analysis. After NMR analysis, each water-soluble web fraction was assayed for inorganic phosphorus (Chen et al., 1956) using KH₂PO₄ as a standard.

Statistical analyses
Pearson correlation coefficients were calculated from molar percentage data: (1) for all pairwise combinations of individual LMM, with data from all non-radioactive web collections from each species pooled for the analyses, (2) between H₂PO ^–₄ and total positively charged LMM, total negatively charged organic LMM, and `excess' positive charge (see `H₂PO ^–₄ and charge balance' in Results), and (3) for all pairwise combinations of web collection number and total readily synthesized, moderately synthesized, and poorly synthesized LMM (see `Synthesis of organic LMM by Argiope' in Results) in web collections from the starving and feeding groups. As in an earlier study (Higgins et al., 2001), all molar percentages were arcsine-transformed prior to analysis and Bonferroni-corrected P values are reported.

Linear regression analysis was used to evaluate changes in three quantities over the series of web collections for a group: (1) the percentage of web mass that was solubilized by water, (2) the percentage of the water-soluble fraction's mass that was accounted for by the 11 organic LMM in A. cavaticus (see `<sup>1</sup>H-NMR analysis' above), and (3) the `excess' positive charge. A t-test was used to determine if slopes of regressions differed significantly from zero. Slopes of `excess' positive charge regressions were compared using a Tukey's multiple comparison test (Zar, 1999).

Specific radioactivities of LMM from radiolabeled webs were normalized as described in Table 1, log₁₀-transformed, and compared using analysis of variance (ANOVA) and Tukey's HSD multiple comparisons test. The specific radioactivities reported in Table 1 are back-transformed means and 95% confidence intervals.

Transformations and slope comparisons were done in Microsoft Excel 2002 (Redmond, WA, USA); correlations, regressions, ANOVAs and multiple comparisons of means were carried out in Systat 10 (Point Richmond, CA, USA).

	Results

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
Synthesis of organic LMM by Argiope
Relative specific radioactivities of the organic LMM from the labeled web collections are presented in Table 1, along with the molar percentages of the organic LMM in these webs. Put and free Pro were not detected by NMR in any of the radiolabeled web collections. Thus, specific activity data were not obtained for these compounds, though the specific activity of the Put component of NAP was measured. The specific activity of free Ala was determined only from the A. trifasciata web extracts because of the very small amounts of Ala in the A. aurantia extracts. Specific activities for Bet and ¹⁴C-labeled Tau were not obtained because we were not convinced these compounds had been adequately resolved. However, relative specific activities were determined for ³⁵S-labeled Tau and for the Tau moiety of ¹⁴C-labeled NAT.

In ¹⁴C-labeled webs, the specific activity of NAT was significantly higher than that of Ise (Table 1). Hydrolysis of NAT, however, revealed that the bulk of the ¹⁴C incorporated into this compound was restricted to its acetyl group and that the specific activities of Ise and the Tau moiety of NAT were not significantly different. Neither were the specific activities of NAT and Ise significantly different in ³⁵S-labeled webs. In contrast to NAT, the label in NAP was more evenly divided between the acetyl and Put moieties, though on a per mole carbon basis, the acetyl group's specific activity was again higher than that of Put, though not significantly so. This difference between the two N-acetylated compounds was in greater measure due to the significantly higher specific activity of the acetyl group in NAT as compared with the acetyl group in NAP, and to a lesser extent due to the higher (though not significantly so) specific activity of the Put moiety as compared with the Tau moiety. Recall that comparisons between these two LMM could be made only in A. trifasciata since NAP was not detected in A. aurantia.

Based on the specific radioactivities obtained, we divided the LMM in Table 1 into three categories: readily synthesized LMM (Ala, Gly, GAB), moderately synthesized LMM (NAP, Put, NAT, Tau, Ise), and poorly synthesized LMM (Cho, Bet). Assuming the acetyl moieties of NAP and NAT can be readily synthesized, LMM with mean specific activities greater than that of the acetyl group of NAP were taken to be readily synthesized. As we did not obtain specific activities for Pro, its placement is uncertain, but it is likely at least a moderately synthesized compound. Free Put and Tau were assumed to have specific activities comparable to those of the Put and Tau moieties of NAP and NAT, respectively, and it was the data for these moieties that dictated the inclusion of NAP and NAT in the moderately synthesized category. Though we also did not obtain data for Bet, we tentatively consider it a poorly synthesized LMM, given earlier observations (Higgins and Rankin, 1999) and the likelihood of its synthesis from Cho (see Discussion).

We predicted that, with starvation, molar percentages of readily synthesized LMM would tend to increase while those of poorly synthesized LMM would decrease and these trends would be reversed when feeding was resumed. The fate of moderately synthesized LMM was more uncertain, but our suspicion was that their molar percentages would either decrease or just be maintained with starvation.

Molar percentages of organic LMM were measured in the radiolabeled web collections simply to allow us to express radioisotope incorporation in terms of relative specific radioactivities. We assumed LMM compositions in these webs would be similar to those in some of the non-radioactive web collections. In some respects they were, but there were also differences. The molar percentages of Cho and Bet are especially noteworthy in this regard. The lowest molar percentage of Cho in the eight radiolabeled A. aurantia web extracts (10.9 mole%) was higher than the highest molar percentage of Cho in the 102 unlabeled A. aurantia web extracts (8.9 mole%). (Note that these two molar percentages were calculated based on the 11 organic LMM constituting 100% of the LMM. The highest molar percentage for Cho in A. aurantia given in Table 2 is 7.4 mole% because the percentages in that table were calculated based on the 11 organic LMM plus H₂PO₄^– constituting 100% of the LMM.) Likewise, the lowest molar percentage of Cho in the three labeled A. trifasciata web extracts (22.6 mole%) was higher than the highest molar percentage of Cho in the 123 unlabeled A. trifasciata web extracts (13.9 mole%). The same sort of discrepancy, albeit not as extreme, was also observed in A. aurantia with regard to molar percentage of Bet. Unlabeled webs were built by adult female Argiope maintained on a diet of flies. They were collected as juveniles and raised in the laboratory through 1–2 molts and were unmated. Labeled webs were built by adult female Argiope that had recently been feeding in the field or, if fed in captivity, were given crickets or grasshoppers. They were collected as adults and were likely mated.

Effects of starvation and web removal on orb web mass and LMM composition
The number of webs composing each web collection is given in supplementary material Table S1.

Gravimetric data
Mass per orb web
Withholding prey and removing webs resulted in an immediate drop in mean orb web mass in four of the five starving groups and the downward trend continued until feeding was resumed (Fig. 1, supplementary material Figs S3–S5). Only in the Argiope aurantia starving adult group was there a slight delay before web mass likewise declined (supplementary material Fig. S6). When feeding was resumed, web mass quickly increased, though, among adults, Argiope trifasciata were slower to recover web mass than A. aurantia or Araneus cavaticus. Among A. cavaticus, juvenile males and females were slower to recover web mass than adult females. Web mass among resumed feeding adults, but not juveniles, ultimately returned to or exceeded the mass at the start of the stadium.

View larger version (59K): [in this window] [in a new window]   Fig. 1. Absolute (A,B) and relative (C,D) quantities of LMM in water extracts of webs built by Argiope trifasciata adult females in feeding (A,C) and starving/resumed feeding (B,D) groups. Average desiccated web masses within web collections are indicated by line graphs in A and B (right y-axis). The key (Bii) identifies the LMM, arranged in the same order, represented in all six bar charts. Several major LMM are also directly labeled in Di to further aid orientation. LMM with a net charge are represented by solid black or white, while net neutral LMM are shown in color. Proline was not detected in any A. trifasciata web collections. GAB, GABamide; Gly, glycine; Ise, isethionate; NAP, N-acetylputrescine.

Web mass as a percentage of spider mass
Table 3 presents data on fresh masses of the three species of spiders and their dry masses fractions. It also presents desiccated masses of webs built by feeding group spiders and the total masses of the 12 measured LMM in these webs. From these data we estimate that these spiders typically invested about 1–3% of their dry mass into the materials composing an orb web and about 0.5–1% of their dry mass into the 12 measured LMM per web.

Water-soluble percentages
For A. cavaticus, the percentage of the desiccated web mass extracted by water (mean ± s.d.) was 71.0±4.24%, N=86 web collections (juveniles 72.4±4.06%, N=51; adults 68.9±3.57%, N=35). For A. aurantia and A. trifasciata, as observed previously (Townley et al., 1991), water-soluble percentages were usually lower, means ± s.d. 54.6±5.70% (N=105) and 52.8±7.45% (N=123), respectively. Water-soluble fractions undoubtedly include some non-LMM components, such as nodular glycoprotein (see Introduction). Very low water-soluble percentages, far below the averages, were seen in some web collections built at or near the end of the series in the A. trifasciata feeding, resumed feeding, PES2 and PES3 groups, and in the A. aurantia PES1 group. It is likely that additional examples of very low water-soluble percentages were masked as a result of end-of-series `horizontal' pooling (see `Gravimetric measurements of orb webs' in Materials and methods and supplementary material Table S1).

Water-soluble percentages tended to decrease over the series. Slopes of linear regressions differed significantly from zero in 14 of the 21 groups, and these 14 slopes were all negative (range: –0.22 to –1.65) and spread across all species and treatments (supplementary material Table S3).

The percentage of the water-soluble mass accounted for by the 11 organic LMM in A. cavaticus was 57.8±5.4% (mean ± s.d., N=86). There was a tendency for this percentage to decrease over the series irrespective of treatment, but linear regression slopes departed significantly from zero in only three of the nine groups of A. cavaticus (feeding juvenile females, P=0.005; starving adult females, P=0.030; resumed feeding adult females, P=0.030). When the data from all nine groups were pooled, the slope of the regression was significantly different from zero (P=0.039), with an average decrease from one web collection to the next of 0.27%.

LMM compositional data
Molar percentage data (averages and ranges) for the 11 organic LMM and inorganic phosphate in the non-radioactive webs of each species are summarized in Table 2. These data were also analyzed for correlations between LMM (Table 4). Some significant correlations showed agreement among our three species and/or with Nephila clavipes L. (Higgins et al., 2001) and these are indicated in Table 4.

Changes in molar percentages of eight of the organic LMM with starvation and subsequent resumed feeding are shown in Figs 2, 3, 4. Fig. 2 contains the readily synthesized LMM, Ala, Gly, and GAB, Fig. 3 the three most abundant moderately synthesized LMM, NAP, NAT and Ise, and Fig. 4 the poorly synthesized LMM, Cho and Bet (see `Synthesis of organic LMM by Argiope' above). These figures also present the corresponding data from the feeding groups.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Molar percentages of GABamide (GAB), free glycine (Gly) and free alanine (Ala) in water extracts of webs built by (A) feeding and (B) starving/resumed feeding groups. In this and Figs 3, 4, a data point representing horizontally pooled web collections, e.g. web collections 14–19, was assigned an x-coordinate midway in this range, i.e. web collection 16.5 (see Materials and methods for explanation of `horizontal pooling' and supplementary material Table S1). Note the very different molar percentage ranges for different compounds. Purple diamonds, juvenile male Araneus cavaticus; green triangles, juvenile female A. cavaticus; red circles, adult female A. cavaticus; blue squares, adult female Argiope aurantia; open circles, adult female Argiope trifasciata.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Molar percentages of N-acetylputrescine (NAP), isethionate (Ise), and N-acetyltaurine (NAT) in water extracts of webs built by (A) feeding and (B) starving/resumed feeding groups. Purple diamonds, juvenile male Araneus cavaticus; green triangles, juvenile female A. cavaticus; red circles, adult female A. cavaticus; blue squares, adult female Argiope aurantia; open circles, adult female Argiope trifasciata.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Molar percentages of choline (Cho) and glycine betaine (Bet) in water extracts of webs built by (A) feeding and (B) starving/resumed feeding groups. Purple diamonds, juvenile male Araneus cavaticus; green triangles, juvenile female A. cavaticus; red circles, adult female A. cavaticus; blue squares, adult female Argiope aurantia; open circles, adult female Argiope trifasciata.

In all tables and figures except Table 1, molar percentages were calculated based on the 12 LMM constituting 100% of the LMM. (In Table 1, molar percentage was calculated based on the 11 organic LMM constituting 100% of the LMM.) In all three species, however, several as-yet-unidentified and generally minor organic LMM were detected in many of the spectra. There are undoubtedly other inorganic LMM components as well, such as NO ^–₃, K⁺, Na⁺ and Ca²⁺ reported from webs of other araneoid species (Schildknecht et al., 1972; Work, 1981; Tillinghast and Christenson, 1984; Patel and Nigam, 1996).

Organic LMM
We predicted that molar percentages of Ala, Gly and GAB would increase with starvation and then drop when feeding was resumed. Conforming to expectations, percentages of Gly and GAB increased with starvation in all three species, at least in the earlier part of the series (Fig. 2). It is especially noteworthy that not only molar percentages, but also absolute quantities of Gly and GAB increased in the webs of starving spiders; Gly in all three species and GAB in Argiope trifasciata, again, at least in the first portion of the series (Fig. 1, supplementary material Figs S3–S6 and Tables S4–S6). With resumed feeding, molar percentages of Gly immediately dropped in all three species, as did GAB in A. trifasciata, though, unexpectedly, they again tended to increase as the resumed feeding series progressed. Other trends were also not expected: free Ala remained a minor component in starving Araneus cavaticus and Argiope aurantia, increasing only slightly (indeed, less than in feeding groups), and in A. trifasciata Ala clearly declined with starvation (Fig. 2). Also, GAB did not decrease with resumed feeding in A. cavaticus and A. aurantia. Even more unanticipated, increases in Gly and GAB were also seen in the feeding groups, where more stable compositions had been expected. These increases are reflected in positive correlations in feeding groups between web collection number and the summed molar percentages of the three readily synthesized LMM, very similar to correlations seen in the starving groups (Table 5).

We suspected that the moderately synthesized LMM would decrease or at most be maintained in starving spiders. The low specific activities of Ise and NAT (Table 1) made it especially likely that they would decline. With starvation we observed trends toward decreased molar percentages of not only Ise and NAT, but NAP as well (Fig. 3). In starving A. cavaticus females, however, decreases in NAT were preceded by increases such that percentages of NAT at the end of the series were similar to those at the beginning. Starving spiders responded to resumed feeding with increased molar percentages of NAP, NAT and Ise, though subsequent trends varied as the resumed feeding series progressed and include examples of declines in molar percentages after the initial rise (e.g. Ise in all three species) as well as more prolonged upward trends (e.g. NAT in A. trifasciata and A. cavaticus). Interestingly, in contrast to the rapid and substantial recovery of Ise in resumed feeding A. trifasciata, resulting in high molar percentages of Ise relative to feeding group A. trifasciata, NAT's recovery was slow and modest, resulting in low percentages of NAT relative to the feeding group (Figs 1, 3).

In agreement with expectations, molar percentages of NAP were essentially maintained by feeding spiders and, while not necessarily maintained at a constant level, percentages of NAT in webs of feeding spiders were generally higher than in the corresponding webs of starving spiders. Ise, however, exhibited molar percentage declines in webs of feeding spiders that were sometimes very similar to those of starving spiders. These contributed to negative correlations between web collection number and summed molar percentages of the moderately synthesized LMM in feeding groups, though these correlations were less significant than those of the starving groups (Table 5). Negative correlations between total readily synthesized and moderately synthesized LMM, and positive correlations between moderately synthesized and poorly synthesized LMM, in both starving and feeding groups, indicate that the moderately synthesized LMM generally exhibit shifts in relative abundance more like poorly synthesized, rather than readily synthesized, LMM (Table 5). Several correlations between NAT and other LMM (Table 4), however, demonstrate that this is not invariably the case (though Table 4, unlike Table 5, considers data from resumed feeding and PES groups as well as from starving and feeding groups).

Cho and Bet were expected to decline with starvation and, indeed, declines were observed in all three species (Fig. 4). But as with GAB, Gly, and Ise, similar trends were also unexpectedly exhibited by the feeding groups (Table 5). Moreover, in the starving groups, after a steep initial decline, Cho tended to level off (or partially rebound) at molar percentages that exceeded the percentages of Cho in the corresponding feeding group webs. In some instances, even µg Cho web^–1 was greater in starving groups than in the corresponding feeding group webs (supplementary material Figs S3–S6 and Tables S4, S5). This contrasted with what we generally observed: greater absolute quantities of LMM per web (often considerably so) in feeding groups than in starving groups due to the usually greater total web mass (Fig. 1, supplementary material Figs S3–S6) and mass of water-soluble fractions in feeding groups. In both Argiope species, negative correlations between web collection number and Cho + Bet were not significant in starving groups (Table 5) primarily due to unexpected end-of-series increases in Cho and Bet. In starving A. cavaticus groups (juveniles especially), Bet molar percentage decreases occurred only after initial unanticipated increases (Fig. 4). Comparable increases were not seen in feeding A. cavaticus. Also contrary to expectations, resumed feeding of starving spiders yielded little or no resurgence in Cho and Bet.

Of the remaining three organic LMM measured in this study, Put and Tau were invariably minor components (<2 mole%) and free Pro's contribution exceeded 2 mole% only in some A. cavaticus webs (Table 2). Each went undetected in all webs from one of the three study species, though it may be that small quantities were not detected because of overlapping peaks from other compounds, including minor unidentified compounds. It now appears that the compound identified as Tau in earlier analyses of A. cavaticus webs (Townley et al., 1991) was not Tau but a minor as yet unidentified compound. Shifts in Pro were reminiscent of Gly with upward trends in both starving and feeding A. cavaticus, and a drop when feeding was resumed by starving spiders (supplementary material Figs S3–S5). Absolute quantities of Pro per web were generally maintained or increased in starving A. cavaticus (supplementary material Table S4).

Post-egg-sac (PES) webs
The construction of up to three egg sacs by some Argiope aurantia and Argiope trifasciata and the building of orb webs after each of these gave us the opportunity to examine changes in LMM composition in webs built between egg laying episodes by feeding spiders (though, as throughout this study, determining LMM composition meant that spiders were not allowed to recycle webs). Cyclical changes in LMM molar percentages were evident when the data from successive PES groups were examined (Fig. 5; absolute quantities of LMM shown in supplementary material Fig. S7). To mention just two examples, in both species Gly was relatively low following egg sac construction, but increased subsequently and then declined with the approach of the next oviposition. Cho, on the other hand, was highest in the first webs built after an egg sac was made and tended to decline as the series progressed.

View larger version (58K): [in this window] [in a new window]   Fig. 5. Changes in LMM composition in webs built after construction of first (PES 1), second (PES 2), and third (PES 3) egg sacs by Argiope aurantia (A) and Argiope trifasciata (B). The PES 3 series from A. aurantia had to be ended prematurely (see Materials and methods). The key identifies the LMM, arranged in the same order, represented in the bar charts. Note that some LMM were either not detected in individual web collections or too minor to be visible in these plots. Glycine betaine and taurine, though not clearly visible in the bar charts, have been retained in the key as a reminder that these LMM were detected in some of these web collections and for consistency with Fig. 1 and supplementary material Figs S2–S7. GAB, GABamide; Gly, glycine; Ise, isethionate; NAP, N-acetylputrescine; NAT, N-acetyltaurine; PES, post-egg-sac group.

H₂PO ^–₄ and charge balance
In the bar charts in Figs 1, 5 and supplementary material Figs S3–S7, LMM carrying a net positive charge at the pH of the sticky droplets [about 4 in Araneus diadematus Clerck 1757 (Schildknecht et al., 1972); 7.9 in Argiope anasuja Thorell 1887 (Patel and Nigam, 1996)] are grouped together, as are net negative and net neutral LMM.

When the molar percentages of the positively charged organic LMM (Put, Cho, GAB and NAP) were added (doubling Put because of its +2 charge) and compared with the sums of the negative organic LMM (NAT and Ise) there was an `excess' of positive charge in all 311 web collections analyzed during this study. This excess was greatest in Argiope aurantia (43.6±0.70 mole%, mean ± s.e.m.; N=102) and least in Argiope trifasciata (21.5±0.65 mole%; N=123), with Araneus cavaticus intermediate (31.0±0.86 mole%; N=86). Analyzing the data from each group separately revealed a highly significant (P0.001) increase over the series in the excess positive charge in all five starving groups and these increases were greater than and significantly different (P0.05) from increases seen in any other groups. In A. cavaticus, there was no significant difference between the starving adult group and either of the starving juvenile groups. Much of the increase in excess positive charge in the starving groups was due to a drop in negatively charged organic LMM (NAT+Ise) over the series rather than to an increase in positively charged organic LMM. In the three starving A. cavaticus groups, >91% of the increased excess positive charge was due to a decrease in NAT+Ise. For the A. aurantia and A. trifasciata starving groups, this percentage was 74% and 48%, respectively.

All web extracts were assayed for inorganic phosphorus to estimate inorganic phosphate's contribution as a counterion to the excess positive charge. In only 9 of the 311 web collections did the molar percentage of H₂PO ^–₄ essentially balance or even slightly exceed the molar percentage of excess positive charge (six of the nine were in the A. trifasciata resumed feeding series and were due primarily to the relatively rapid recovery of Ise near the start of the series decreasing the excess positive charge). In the remaining 302 web collections the molar percentage of H₂PO ^–₄ was insufficient to balance the molar percentage of excess positive charge. On average, the percentage of the excess positive charge balanced by inorganic phosphate was least in the species with the greatest excess positive charge, A. aurantia (36.3%), and about equal in the other two species (A. trifasciata 47.7%, A. cavaticus 48.8%). As noted above, the 12 LMM quantitated in this study do not provide a complete inventory of the LMM, especially of inorganic ions. And a more complete accounting of charges in this system should also consider components other than LMM, such as the nodular glycoprotein (see Introduction).

There was a positive correlation between the molar percentages of H₂PO ^–₄ and excess positive charge when the data from all three species were pooled (r=0.651, P<0.001, N=311), but only A. cavaticus (r=0.546, P<0.001, N=86) and A. trifasciata (r=0.386, P<0.001, N=123) showed significant correlations when each species was analyzed separately. In A. cavaticus, this correlation was attributable to a negative correlation between H₂PO ^–₄ and negatively charged organic LMM (NAT+Ise) (r=–0.643, P<0.001), as opposed to the situation in A. trifasciata in which there were significant correlations between H₂PO ^–₄ and both negatively charged (r=–0.235, P=0.009) and positively charged (r=0.274, P=0.002) organic LMM. The inverse relationship between H₂PO ^–₄ and the two sulfonic acids was mostly clearly exhibited in juvenile A. cavaticus (males and females) and contributed to significant inverse correlations between H₂PO ^–₄ and both NAT and Ise in A. cavaticus (Table 4).

	Discussion

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
Synthesis of sticky droplet organic LMM
Radiolabeled glucose and methionine/cysteine were fed to spiders to assess their ability to synthesize sticky droplet LMM. Label from [¹⁴C]glucose appeared in high specific radioactivity in web droplet Ala, Gly and GAB, while only a meager amount was associated with Cho (Table 1), indicating that Cho is nutritionally essential. Ise, Tau (free and as a component of NAT) and the Put moiety of NAP occupied positions between these extremes, with the relatively low specific activities of the two sulfonic acids in particular raising the possibility that, without adequate dietary intake of these LMM (or more immediate precursors), the spider's synthetic rate may not always be sufficient to meet the requirements for optimal web construction. These results are essentially in agreement with the literature, as detailed in the following sections.

GABamide, glycine and alanine (readily synthesized LMM)
Relatively high specific activities for Ala, Gly and GAB are in keeping with previous studies on Araneus cavaticus fed [¹⁴C]glucose (Townley and Tillinghast, 1988) and Nephila clavipes fed [¹⁴C]glucose or [¹⁴C]acetate (Higgins and Rankin, 1999). That Gly and Ala were readily synthesized is not surprising as these amino acids are generally dispensable in arthropods (Dall and Moriarty, 1983; Dadd, 1985; Guillaume, 1997). Nor was it surprising that in each of the three ¹⁴C-labeled Argiope trifasciata web collections the specific activity of Ala was higher than that of Gly. Such a disparity in animals administered [U-¹⁴C]glucose has often been observed, including in other arachnids (Rodriguez and Hampton, 1966; Rodriguez and Lasheen, 1971), insects (see references in Kasting and McGinnis, 1966; also Rock and Hodgson, 1971; Widmer, 1973) and crustaceans (e.g. Shewbart et al., 1972; van Marrewijk and Zandee, 1975; Lasser and Allen, 1976; Miyajima et al., 1976).

In an earlier study with N. clavipes webs (Higgins et al., 2001), molar percentages of free Gly and Ala tended to be positively correlated. Likewise, in this study, Gly and Ala were positively correlated in the three species and, in A. cavaticus and Argiope aurantia, Pro was positively correlated with both Gly and Ala (Table 4) (Pro was not detected in webs of A. trifasciata).

N-Acetyltaurine, isethionate and N-acetylputrescine (moderately synthesized LMM)
Earlier studies examined the incorporation of radioisotope into LMM from webs built by Argiope fed [¹⁴C]Tau or [³⁵S]cysteine (Anderson and Tillinghast, 1980; Tillinghast, 1984). However, as those studies preceded the identifications of NAT and Ise (Vollrath et al., 1990), only tentative conclusions may be drawn from them. Nevertheless, it appeared that, after feeding either radioactive precursor, labeled NAT (`compound I' of Anderson and Tillinghast, 1980), Ise (consistent with `compound II'), and Tau were present in webs built by A. trifasciata, while in A. aurantia only NAT was unambiguously labeled. This difference may have been due to different relative quantities of sulfonates on the webs: A. aurantia webs typically contain lower molar percentages of Ise and higher NAT than A. trifasciata webs (Vollrath et al., 1990; Townley et al., 1991) (Table 2). In webs of N. clavipes, Ise has not been detected by NMR (Higgins et al., 2001), but radiolabeling of NAT by spiders fed [¹⁴C]glucose or [¹⁴C]acetate has been observed, albeit inconsistently (Higgins and Rankin, 1999). Results obtained to date from Argiope fed [³⁵S]cysteine (Anderson and Tillinghast, 1980) (Table 1) indicate that these spiders can, like many other animals (Jacobsen and Smith, 1968; Allen and Garrett, 1971; Chen, 1985; Huxtable, 1986), synthesize Tau from cysteine, though we have no information on which pathway(s) is(are) used in this conversion.

The incorporation of radioisotope into Ise by Argiope fed [¹⁴C]Tau (Anderson and Tillinghast, 1980) needs verification, but if true would suggest that Ise is formed from Tau. Though this conversion apparently occurs in some organisms (Braun and Fromageot, 1962), it has not been established that Ise synthesis from Tau occurs in animal tissues (Huxtable, 1986; Fellman, 1987). More recent studies with human neutrophils have again raised the possibility of a Tau to Ise conversion in animals (Cunningham et al., 1998; Cunningham and Tipton, 2000), but the evidence is inconclusive.

Given the comparable specific activities obtained for Ise and the Tau moiety of NAT in the present study (Table 1), one possible alternative pathway to Ise (Cavallini et al., 1978; Scandurra et al., 1978) warrants mention. In this pathway, cystamine would give rise to equal amounts of cysteamine and 2-mercaptoethanol, from which the former can be converted to Tau (Cavallini et al., 1976; Huxtable and Bressler, 1976; Read et al., 1976) and the latter to Ise (Federici et al., 1976; Dupré et al., 1978). Here, Tau is not a precursor, but a coproduct of the pathway to Ise.

NMR analyses have not revealed NAP in webs of N. clavipes, but Put is present in quantity (Higgins et al., 2001), as it is in some other araneoids [e.g. Metepeira incrassata F.O.P.-Cambridge 1903 (Higgins et al., 2001), Micrathena gracilis (Walckenaer 1805) (M. A. Townley and E. K. Tillinghast, unpublished)]. Put was radiolabeled in some webs built by N. clavipes fed [¹⁴C]glucose or [¹⁴C]acetate, but not as consistently as GAB, Gly, and Ala (Higgins and Rankin, 1999). Likewise, in this study the specific activity of the Put component of NAP was significantly lower than the specific activities of GAB, Gly and Ala (Table 1).

Choline and glycine betaine (poorly synthesized LMM)
In earlier studies, radioisotope was not observed in Cho from webs built by A. aurantia fed [¹⁴C]glucose (Tillinghast and Townley, 1994) or N. clavipes fed [¹⁴C]glucose or [¹⁴C]acetate (Higgins and Rankin, 1999). Insects, likewise, are either unable to synthesize Cho or synthesize insufficient Cho (via methylation of phosphatidylethanolamine), making it a nutritional essential (Dadd, 1985). Limited synthesis of Cho from phosphatidylethanolamine also occurs in at least some crustaceans (Bilinski, 1962; Shieh, 1969; Brichon et al., 1980; D'Abramo and Baum, 1981), but again most evidence suggests that crustaceans have a dietary requirement for Cho (D'Abramo and Baum, 1981; Catacutan and de la Cruz, 1989; Kanazawa et al., unpublished; cited in Boonyaratpalin, 1996; Shiau, 1998; Shiau and Lo, 2001). Indeed, Cho's essential status may apply to arthropods generally (Morris, 1991).

In the present study, the specific radioactivity of Bet was not determined because of suspected contamination, but our observations indicate that labeling of this compound was slight at most. An earlier study found that Bet from webs of N. clavipes fed [¹⁴C]glucose or [¹⁴C]acetate was not radiolabeled and that this compound is likely essential (Higgins and Rankin, 1999). A significant positive correlation between molar percentages of Bet and Cho was reported in one population of N. clavipes (Higgins et al., 2001) and was likewise observed in all three species used in this study (Table 4). Such observations are consistent with Bet synthesis from Cho via betaine aldehyde, to our knowledge the only established pathway to Bet in both chelicerate (Dragolovich and Pierce, 1994) and mandibulate (Bilinski, 1960; Weiher and Komnick, 1997) arthropods.

Is there agreement between synthetic capacity and LMM compositional changes with starving?
Assuming an organic LMM's specific radioactivity is a reliable indicator of the spider's ability to synthesize that LMM, we anticipated that, with fasting, there would be decreased molar percentages of those LMM showing lower incorporation of radioisotope. Was this expectation met? In some respects, yes. Downward trends in starving spiders were most evident among `poorly synthesized' (Cho, Bet) and `moderately synthesized' (Ise, NAT, NAP, Put) LMM, with the exception of the decline in Ala in A. trifasciata. Extended upward trends were seen only among `readily synthesized' LMM (GAB, Gly and, to a far lesser extent, Ala in A. cavaticus) and Pro (presumed at least a moderately synthesized LMM). Even more than increases in molar percentage, increases in µg web^–1 of Gly, GAB and Pro, as seen over at least part of a starving series in one or more species, suggest that starving spiders use these LMM to compensate to some extent for decreases in less readily synthesized and less available LMM.

Argiope trifasciata provided a particularly striking example of a compositional shift accompanying starvation that may have resulted from differences in synthetic capacity; specifically, the decline in molar percentage of NAP and coincident increase in GAB. In webs of this species, the mean specific radioactivity of GAB was almost four times higher than that of the Put moiety of NAP (Table 1). Unlike webs of A. cavaticus and A. aurantia, webs of A. trifasciata contain NAP as a major constituent whereas GAB is generally less abundant (Townley et al., 1991) and sometimes even a minor component, as at the start of the feeding and starving series (Figs 1, 2). This suggests that these two similar compounds may fulfill the same, as yet un