Small ampullate glands of Nephila clavipes

The small ampullate glands of the orb-web spider, Nephila clavipes, have been studied and compared to other of the silk producing glands from this organism. They exhibit the same gross morphological features of the other glands. Electrophoretic analyses show that the gland's luminal contents migrate as a single band, while the contents of the secretory epithelium reveal a step-ladder array of peptides in addition to the full size product. Previous studies from our laboratory identified these peptides as products generated by translational pauses. This alternate mode of translation is typical of fibroin synthesis in all the spider glands thus far studied as well as in those of the silkworm. The correlation of the peptides to the process of fibroin synthesis is shown through experimental evidence in this paper. The gradual ultrastructural changes in Golgi vesicles elicited by the fibroin synthesis stimulus can be seen in this paper. The response to stimulation is of a higher magnitude in these glands than in any of those previously analyzed. These studies show the small ampullate glands are a promising and certainly exploitable model system for studies on the synthesis of tissue-specific protein product and its control. J. Exp. Zool. 286:114-119, 2000.     

Presence of phosphorus in Nephila clavipes dragline silk.

Solid-state 31P-NMR of Nephila clavipes dragline silk indicates the presence of phosphorus in at least two chemically distinct environments. Amino acid analyses of acid-hydrolyzed silk confirm the presence of phosphotyrosine as one of the phosphorus-containing components. The unusual chemical shift (18.9 ppm downfield from 85% H3PO4), proton chemical shift, and acid lability of a second component suggest that it is part of a strained five-membered cyclic phosphate that might be found on a beta-D-ribose. The five-membered cyclic phosphate is not removed from the silk fibers by exhaustive aqueous extraction. It is absent in nascent silk fibroin from the glands, suggesting that its formation is part of the fiber processing that occurs in the ducts leading to the spinnerets. High-resolution NMR spectra of silk dissolved in propionic acid/12 N HCl (50:50 v/v) show five phosphorus sites assigned to phosphorylated tyrosine residues, phosphorylated serine residues, inorganic phosphate, and two hydrolysis products of the cyclic phosphate compound. The observed posttranslational phosphorylation may be important in the processing and modulation of the physical properties of dragline silk.     

Conspecific Attraction Drives Intraspecific Aggregations by Nephila clavipes Spiders

Aggregation behavior is common throughout the animal world, although it is rare in spiders (Araneae). Nephila clavipes spiders are frequently found both in solitary webs and in aggregations of conspecifics. N. clavipes aggregations are not considered social, because these spiders do not engage in food sharing or group parental care. The currently accepted explanation for N. clavipes aggregations is that they form passively, as spiders aggregate in response to the patchy distribution of food resources in the environment. Here, we show instead that N. clavipes females actively seek out conspecifics and, therefore, that aggregations arise from conspecific attraction. We experimentally established that (1) N. clavipes females actively follow the trails left by conspecifics and (2) aggregations are formed independently of prey density in a particular area. These results suggest that while aggregations in high food patches increase encounter rates of conspecific spiders and support the nutritional needs of multiple large spiders, this potential benefit is not the proximate mechanism for aggregation. Thus, we hypothesize that N. clavipes females likely acquire fitness benefits from aggregating with conspecifics.     

Characterization of the protein components of Nephila clavipes dragline silk

Spider silk is predominantly composed of structural proteins called spider fibroins or spidroins. The major ampullate silk that forms the dragline and the cobweb's frame threads of Nephila clavipes is believed to be a composite of two spidroins, designated as Masp 1 and 2. Specific antibodies indeed revealed the presence of Masp 1 and 2 specific epitopes in the spinning dope and solubilized threads. In contrast, sequencing of specific peptides obtained from solubilized threads or gland urea extracts were exclusively homologous to segments of Masp 1, suggesting that this protein is more abundantly expressed in silk than Masp 2. The strength of immunoreactivities corroborated this finding. Polypeptides reactive against both Masp 1 and 2 specific antibodies were found to be expressed in the epithelia of the tail and different gland zones and accumulated in the gland secreted material. Both extracts of gland secretion and solubilized threads showed a ladder of polypeptides in the size range of 260-320 kDa in gel electrophoresis under reducing conditions, whereas gel filtration chromatography yielded molecular masses of the proteins of approximately 300-350 kDa. In the absence of a reducing agent, dimeric forms of the spidroins were observed with estimated molecular masses of 420-480 kDa according to gel electrophoresis and 550-650 kDa as determined by gel filtration chromatography. Depending on the preparation, some silk material readily underwent degradation, and polypeptides down to 20 kDa in size and less were detectable.     

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all ¹H, ¹³C, and ¹⁵N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes.     

13C NMR of Nephila clavipes major ampullate silk gland.

The major ampullate glands of the spider Nephila clavipes contain approximately 0.2 microliter each of a highly concentrated (approximately 50%) solution of silk fibroin. Therefore, the reservoir of silk in these glands presents an ideal opportunity to observe prefolded conformations of a protein in its native state. To this end, the structure and conformation of major ampullate gland silk fibroin within the glands of the spider N. clavipes were examined by 13C NMR spectroscopy. These results were compared to those from silk protein first drawn from the spinneret and then denatured. The 13C NMR chemical shifts, along with infrared and circular dichroism data, suggest that the silk fibroin in the glands exists in dynamically averaged helical conformations. Furthermore, there is no evidence of proline residues in U-(13)C-D-glucose-labeled silk. This transient prefolded "molten fibril" state may correspond to the silk I form found in Bombyx mori silk. There is no evidence of the final beta-sheet structure in the ampullate gland silk fibroin before final silk processing. However, the conformation of silk in the glands appears to be in a highly metastable state, as plasticization with water produces the beta-sheet structure. Therefore, the ducts connecting the ampullate glands to the spinnerets play a larger role in silk processing than previously thought.     

Nutritional requirements for web synthesis in the tetragnathid spider Nephila clavipes

Trap-constructing organisms provide a unique opportunity for the study of resource allocation, because an observer can unambiguously determine the allocation to foraging. In species that synthesize a trap from physiologically important compounds, there is the further advantage that there may be direct trade-offs between allocation of resources to foraging and physiological functions. We examined the ability of the spider Nephila clavipes (L.; Araneae: Tetragnathidae) to synthesize resources that are known to be used for both web synthesis and non-foraging physiological functions. We found that choline, required for both web function and physiological function, is an essential nutrient: it is not synthesized by this spider. Under laboratory conditions with a diet of fruit flies, choline is limiting, and the spiders make allocation trade-offs between investing choline in foraging (the web) or in their body.     

Discrimination and release of unpalatable butterflies by Nephila clavipes, a neotropical orb-weaving spider

Abstract. 1. Nephila clavipes (L.), a common spider in neotropical forests, discriminates some unpalatable prey and releases them unharmed from its web. Release is not accidental but results from a specific behavioural sequence.
2. Field trials with twenty-seven butterfly species showed that spiders respond consistently to butterfly species and higher taxa. Ithomiinae and some Danainae are almost always released while Heliconiinae, Nymphalinae, Acraeinae, Pieridae and Papilionidae are usually eaten.
3. Paired tests showed that an immediately preceding experience with a different butterfly did not reveal any change in the spider's usual response to a particular butterfly.
4. Warning coloration is not involved in spider response. Spiders rejected the models but ate the mimics of two different butterfly species pairs. Distastefulness is probably signalled by chemical cues.
5. Some unpalatable butterflies stay motionless when entangled and while the spiders release them. Motionlessness in webs seems to be a requisite to allow recognition of their distastefulness without being bitten by the spider.
6. Spiders differ from vertebrates in the prey accepted and in rejection mechanics. Although there is no indication of learning, spiders may select butterflies for distastefulness, chemical signals and motionlessness, at the individual level.     

Variation of mechanical properties with amino acid content in the silk of Nephila clavipes

In this paper, we explore the impact of dietary deprivation, where spiders are provided diets missing one or more of the amino acids, on the properties of the spider dragline silk spun after one month on the diet. Cohorts of female N. clavipes spiders were selected for diets deprived of alanine (Ala) and glycine (Gly), arginine (Arg), leucine (Leu), or tyrosine (Tyr), and their silk was harvested twice weekly during the one-month course of the diet. Significant mechanical differences are observed after as little as 6 days on the diet. Utilizing conventional tensile testing methods, single fibers were strained to break so as to study the influence of diet on the stress/strain properties. Diets deprived of Ala and Gly appear to most directly impact the load-bearing foundation of dragline silk. Diets deprived of Arg, Tyr, and possibly Leu reduce the strength of the silk, and diets missing Tyr and Leu reduce the strain-to-failure. Observations obtained from ESEM photos of the fracture interfaces after tensile testing illustrate the fracture mechanics of spider silk. Both solid-state NMR and amino acid analysis of the digested protein suggest, however, that the relationship between diet and amino acid incorporation into the silk fiber is not straightforward.     

Patterns of variation among distinct alleles of the Flag silk gene from Nephila clavipes

Spider silk proteins and their genes are very attractive to researchers in a wide range of disciplines because they permit linking many levels of organization. However, hypotheses of silk gene evolution have been built primarily upon single sequences of each gene each species, and little is known about allelic variation within a species. Silk genes are known for their repeat structure with high levels of homogenization of nucleotide and amino acid sequence among repeated units. One common explanation for this homogeneity is gene convergence. To test this model, we sequenced multiple alleles of one intron-exon segment from the Flag gene from four populations of the spider Nephila clavipes and compared the new sequences to a published sequence. Our analysis revealed very high levels of heterozygosity in this gene, with no pattern of population differentiation. There was no evidence of gene convergence within any of these alleles, with high levels of nucleotide and amino acid substitution among the repeating motifs. Our data suggest that minimally, there is relaxed selection on mutations in this gene and that there may actually be positive selection for heterozygosity.     

Effects of male size and female dispersion on male mate-locating success in Nephila clavipes

The reproductive success of sexually reproducing organisms depends on their capacity to locate potential partners. Research on the mating systems of web-building spiders, like other terrestrial invertebrates, has focused around male courtship and the signalling behaviours that facilitate copulation. In contrast, both mate searching and mate location behaviours are largely ignored and rarely quantified in field studies. We conducted a series of field removal experiments to explore the effects of male body size and female dispersion on mate locating behaviour of the golden orb-weaving spider (Nephila clavipes). The number of males that arrived on females’ webs was influenced by travel distance, male body size, male body weight and female dispersal. More males returned to webs that were closer to the release point; smaller males experienced a greater weight loss; and larger numbers of males arrived at webs where males had been previously present. Female dispersion influenced the size but not the number of arriving males: males were no more likely to arrive at solitary than aggregations of webs, but males arriving at solitary webs were typically larger than those arriving at aggregations. We discuss the implications of various male traits that could act as selective pressures on male mate location and mate searching behaviours.     

Conformational and orientational transformation of silk proteins in the major ampullate gland of Nephila clavipes spiders

The orientational and conformational transformation of the native liquid silk into a solid fiber in the major ampullate gland of the spider Nephila clavipes has been studied by Raman spectromicroscopy. The spectra show that the conformation of silk proteins in the glandular sac contains several secondary structure elements, which is consistent with intrinsically unfolded proteins. A few alpha-helices are also present and involve some alanine residues located in the polyalanine segments of the spidroin sequence. The conversion of the silk solution in the major ampullate gland appears to be a two-state process without intermediate states. In the first and second limbs of the duct, silk is isotropic and spidroins are generally native-like. beta-Sheets start to develop between the second and the third limb of the duct, suggesting that early beta-sheets are generated by shear forces. However, most of the beta-sheets are formed between the draw down taper and the valve. The early beta-sheets formed upward of the draw down taper might play the role of nucleation sites for the subsequent beta-sheet aggregation. The alignment of the polypeptides chains occurs near the valve, revealing that orientational and conformational changes do not occur simultaneously. Extensional flow seems to be the driving force to produce the orientational order, which in turn is associated with the formation of the major part of the beta-sheets. The slow evolution of the spidroin conformation up to the draw down taper followed by the rapid transformation between the drawn down taper and the valve may be important to achieve the optimal structure of the final fiber.     

Vibrations in the orb web of the spider Nephila clavipes: cues for discrimination and orientation

Transmission of natural and artificial vibrations in webs of Nephila clavipes was examined using laser Doppler vibrometry to determine how this spider discriminates and localizes stimuli. 1. Vibration signals of four entrapped insect species peaked at different frequencies from 5–30 Hz, but their spectra overlapped considerably. Peak amplitudes spanned 50 dB. 2. Transmission of longitudinal vibrations along individual radii was attenuated over ca. 12 cm by 4.0 ± 2.7 dB; attenuation values for transverse and lateral vibrations were 22.2 ± 4.6 dB and 26.2 ± 4.3 dB, respectively. Some transmission spectra characteristics may be explained by resonances of the spider and threads. 3. Radial thread transmission increased by 2.2–5.8 dB after cutting the connecting auxiliary spirals, demonstrating that vibrations leak from stimulated radii via these threads. Auxiliary spirals provide structural support to Nephila webs at the expense of degraded directional transmission. 4. Upon single-point stimulation, vibrations measured around the web hub and at the spider's tarsi revealed 2-D vibration amplitude gradients of 20–30 dB indicating the stimulus direction. In contrast, measured vibration propagation velocities of 70–1500 m/s resulted in time-of-arrival differences at the spiders tarsi of < 1.5 ms, which may be too brief for stimulus direction determination.Abbreviations A area - C propagation velocity - E Young's modulus - LDV laser Doppler vibrometer/-metry - r.h. relative humidity - T tension - space constant - radius of gyration - density - 2-D two-dimensional - 3-D three-dimensional     

An investigation of the divergence of major ampullate silk fibers from Nephila clavipes and Argiope aurantia

The major ampullate fiber of both Nephila clavipes and Argiope aurantia is composed of two different proteins, MaSp1 and MaSp2. Each of these proteins has a highly conserved pattern of silk-associated amino acid motifs. The GPGXX motif is the only source of proline and is unique to MaSp2. On the basis of the percent of proline, Nephila clavipes major ampullate silk was calculated to consist of 19% MaSp2 and 81% MaSp1, while Argiope aurantia was calculated to have a significantly higher MaSp2 content of 59% with MaSp1 comprising the remaining 41%. To investigate the functional implications of the difference in protein composition, major ampullate silk fibers from Nephila clavipes and Argiope aurantia were mechanically tested and compared. Stress-strain curves produced from polynomial regression show that the two significant differences between major ampullate silk fibers from Nephila clavipes and Argiope aurantia are the average peak load stress and Young's modulus, with Argiope higher for both.     

Male contest investment changes with male body size but not female quality in the spider Nephila clavipes

Animals use rules to adjust their level of investment in a contest. We evaluate male strategies during contests over females in the golden orb-web spider Nephila clavipes. We tested whether male behaviour changes with female value, and found that contests were similar in intensity and outcome whether the female was a juvenile or adult, virgin or non-virgin, or whether one male had invested sperm in the female. We found evidence that males use a self-assessment strategy when deciding to withdraw from a contest. Loser body size and contestant size difference were correlated with a higher frequency of contest escalation, and fights involving two large males were more likely to escalate than a fight in which one male was small. A multiple regression showed that loser body size had a stronger effect on contest escalation than contestant body size difference. More importantly, the size of the winning male had no effect on contest escalation, a key prediction of a self-assessment strategy. In N. clavipes, body size is the primary factor that determines the outcome of male contests, and males do not appear to assess their opponent or the quality of the resource when deciding to withdraw from the fight.     

Multiple bradykinin-related peptides from the capture web of the spider Nephila clavipes (Araneae, Tetragnatidae)

Three bradykinin-related peptides (nephilakinins-I to -III) and bradykinin itself were isolated from the aqueous washing extract of the capture web of the spider Nephila clavipes by gel permeation chromatography on a Sephacryl S-100 column, followed by chromatography in a Hi-Trap Sephadex-G25 Superfine column. The novel peptides occurred in low concentrations and were sequenced through ESI-MS/MS analysis: nephilakinin-I (G-P-N-P-G-F-S-P-F-R-NH2), nephilakinin-II (E-A-P-P-G-F-S-P-F-R-NH2) and nephilakinin-III (P-S-P-P-G-F-S-P-F-R-NH2). Synthetic peptides replicated the novel bradykinin-related peptides, which were submitted to biological characterizations. Nephilakinins were shown to cause constriction on isolated rat ileum preparations and relaxation on rat duodenum muscle preparations at amounts higher than bradykinin; apparently these peptides constitute B2-type agonists of ileal and duodenal smooth muscles. All peptides including the bradykinin were moderately lethal to honeybees. These bradykinin peptides may be related to the predation of insects by the webs of N. clavipes.