Small organic solutes in sticky droplets from orb webs of the spider Zygiella atrica (Araneae; Araneidae): ß-alaninamide is a novel and abundant component.

In northeastern North America, Zygiella atrica often build their orb webs near the ocean. We analyzed individual field-built Z. atrica webs to determine if organic low-molecular-mass solutes (LMM) in their sticky droplets showed any unusual features not previously seen in orb webs of other species living in less salty environments. While two of the three most abundant organic LMM (putrescine (butane-1,4-diamine) and GABamide (4-aminobutanamide)) are already well-known from webs of inland spiders, the third major LMM, ß-alaninamide (3-aminopropanamide), a homolog of GABamide, has not been detected in sticky droplets from any other araneoid spiders (27 species). It remains to be established, however, whether or not use of ß-alaninamide is related to proximity to saltwater. We observed variability in organic LMM composition in Z. atrica webs that appeared to be influenced more by an undetermined factor associated with different collecting locations and/or collection dates than by different genders or instars. Shifts in composition when adult females were transferred from the field to the laboratory were also observed. Structural similarities and inverse correlations among ß-alaninamide, GABamide, and N-acetylputrescine suggest that they may form a series of LMM fulfilling essentially the same, as yet unknown, role in the webs of those species in which they occur.

Molt-related changes in the major ampullate silk gland of the barn spider Araneus cavaticus.

Spiders molt periodically before reaching full maturity, but several spiders continue to molt after sexual maturity. This post-maturity molting (PMM) behavior has been observed in the barn spider Araneus cavaticus (Araneae: Araneidae) among the orb-web spiders. In this study, we investigated molt-related changes in the ampulla and tail regions of the major ampullate gland during the PMM sequences (intermolt, pre-molt, ecdysis, and post-molt). The results showed that all gland units consist of a monolayer of epithelial cells surrounding a large central lumen, and two types of secretory granules (Type-M and Type-S). During the molting period, most cells showed fine structural modification in their organelles, and conspicuous tissue swelling was detected at the glandular epithelium. Following the molting cycle, the amount of Type-M granules continues to increase in the cell with a corresponding swelling, but Type-S granules gradually disappeared during the process of ecdysis. This suggests that the molt-related changes in spider silk production originates from the periodic production of Type-S secretory granules in the ampulla region. As Type-M granules flow toward the funnel, it is coated with viscous liquid secretion of Type-S granules in order to produce dragline silk fibers. We provide fine structural evidence for Type-S granules of hexagonal crystalline substructures representing glycoprotein substances to maintain high level of water content.

Free amino acids in spider hemolymph.

We examined the free amino acid composition of hemolymph from representatives of five spider families with an interest in knowing if the amino acid profile in the hemolymph of orb-web-building spiders reflects the high demands for small organic compounds in the sticky droplets of their webs. In nearly all analyses, on both orb and non-orb builders, glutamine was the most abundant free amino acid. Glycine, taurine, proline, histidine, and alanine also tended to be well-represented in orb and non-orb builders. While indications of taxon-specific differences in amino acid composition were observed, it was not apparent that two presumptive precursors (glutamine, taurine) of orb web sticky droplet compounds were uniquely enriched in araneids (orb builders). However, total amino acid concentrations were invariably highest in the araneids and especially so in overwintering juveniles, even as several of the essential amino acids declined during this winter diapause. Comparing the data from this study with those from earlier studies revealed a number of discrepancies. The possible origins of these differences are discussed.

Astacin family metallopeptidases and serine peptidase inhibitors in spider digestive fluid.

Digestive fluid of the araneid spider Argiope aurantia is known to contain zinc metallopeptidases. Using anion-exchange chromatography, size-exclusion chromatography, sucrose density gradient centrifugation, and gel electrophoresis, we isolated two lower-molecular-mass peptidases, designated p16 and p18. The N-terminal amino acid sequences of p16 (37 residues) and p18 (20 residues) are 85% identical over the first 20 residues and are most similar to the N-terminal sequences of the fully active form of meprin (beta subunits) from several vertebrates (47-52% and 50-60% identical, respectively). Meprin is a peptidase in the astacin (M12A) subfamily of the astacin (M12) family. Additionally, a 66-residue internal sequence obtained from p16 aligns with the conserved astacin subfamily domain. Thus, at least some spider digestive peptidases appear related to astacin of decapod crustaceans. However, important differences between spider and crustacean metallopeptidases with regard to isoelectric point and their susceptibility to hemolymph-borne inhibitors are demonstrated. Anomalous behavior of the lower-molecular-mass Argiope peptidases during certain fractionation procedures indicates that these peptidases may take part in reversible associations with each other or with other proteins. A. aurantia digestive fluid also contains inhibitory activity effective against insect digestive peptidases. Here we present evidence for at least thirteen, heat-stable serine peptidase inhibitors ranging in molecular mass from about 15 to 32 kDa.

Relation between the outer cover of the egg case of Argiope aurantia (Araneae: Araneidae) and the emergence of its spiderlings.

To emerge from the egg case, Argiope aurantia spiderlings must penetrate a tightly woven outer cover composed primarily of large-diameter cylindrical gland fibers and small-diameter fibers, likely of aciniform gland origin. They accomplish this using enzymatic digestion and mastication to form a communal hole in the outer cover. The involvement of proteolytic enzymes in this process was demonstrated by zymography of spiderling homogenates and washes made from the edges of holes. The specific source(s) of the proteases is unknown, but histological examination of spiderling sections indicates that the digestive tract, venom glands, and gnathocoxal glands are all functioning at the time of emergence from the egg case. Observations on edges of holes indicate that spiderlings are able to solubilize the small-diameter fibers completely, but cylindrical gland fibers only partially. In the outer cover, cylindrical fibers are composed of numerous fibrils embedded within a matrix. Spiderlings appear to be unable to solubilize the fibrils, but digestion of the matrix allows the spiderlings to push the fibrils aside to create the opening.

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

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.

Fibrous and adhesive components of the orb webs of Araneus trifolium and Argiope trifasciata.

The minimum number of secretion products used by the spiders Araneus trifolium and Argiope trifasciata to construct their orb webs has been established by selective enzyme digestion and histochemical staining, as well as differential isotope localization in these webs. Three fiber types are present in the webs: (a) a major fiber found throughout the web, (b) a minor fiber found only in radial threads, and (c) the core fibers of the sticky spiral thread. Three nonfibrous secretions are found on these fibers. These include a water soluble viscid coating of the sticky spiral and two adhesives which fasten the threads of the web together; one found only at junctions of sticky spiral and radial threads and the other at all other thread connections. The possible glandular sources of these secretions are discussed.

Carbohydrates in the webs of Argiope spiders.

Glycoproteins are present in the web of the orb-weaving spiders Argiope trifasciata and Argiope aurantia. Periodic acid-Schriff reactive glyco-proteins are confined in large part, to the sticky spiral and sticky spiral-radial junctions. Glycoproteins containing amino sugars appear associated with all fibers, especially the radial fibers. Enzymes may be used to remove glycoproteins selectively from the sticky spiral and stabilimentum.

Selected aspects of spinning apparatus development in Araneus cavaticus (Araneae, Araneidae).

In the first half of this century, several workers observed small, seemingly glandular structures attached to the ampullate glands of spiders. Hence, they were termed accessory ampullate glands. In juvenile Araneus cavaticus, two pairs of these structures are present (starting at least with third instars), one pair attached to the major ampullate (MaA) glands and the other pair attached to the minor ampullate (MiA) glands. In adults, two pairs of accessory MaA glands and two pairs of accessory MiA glands are present. The two latter-formed pairs of accessory ampullate glands are clearly the remnants of those ampullate glands which atrophy shortly after adulthood is reached. Morphological similarities between these accessory ampullate glands and those present in juveniles provide an indication that the latter also have their origin in functional ampullate glands. A reduction in the number of ampullate glands following the last molt occurs in many spiders. The reason(s) for these reductions is unknown. In penultimate spiders close to ecdysis, we have observed that while the larger pairs of MaA and MiA glands (those that are retained in the adult) are undergoing molt-related changes which apparently render them nonfunctional, their smaller counterparts are seemingly unaffected and functional. This raises the possibility that the principal role of the smaller ampullate glands may be to assume functions during the pre-ecdysial period which are normally in the domain of the larger ampullate glands. If true, then their degeneration after the last molt would make economic sense. The presence of cylindrical spigots in juvenile females starting with fourth instars is documented.