Special Prey, Special Glue: NMR Spectroscopy on Aggregate Glue Components of Moth-Specialist Spiders, Cyrtarachninae

Abstract

Orb-weaver spiders produce upwards of seven different types of silk, each with unique material properties. We focus on the adhesive within orb-weaving spider webs, aggregate glue silk. These droplets are composed of three main components: water, glycoproteins, and a wide range of low molecular mass compounds (LMMCs). These LMMCs are known to play a crucial role in maintaining the material properties of the glycoproteins, aid in water absorption from the environment, and increase surface adhesion. Orb-weavers within the Cyrtarachninae subfamily are moth specialists and have evolved glue droplets with novel material properties. This study investigated the biochemical composition and diversity of the LMMCs present in the aggregate glue of eight moth-specialist species and compared them with five generalist orb-weavers using nuclear magnetic resonance (NMR) spectroscopy. We hypothesized that the novel drying ability of moth-specialist glue was accompanied by novel LMMCs and lower overall percentages by silk weight of LMMCs. We measured no difference in LMMC weight by the type of prey specialization, but observed novel compositions in the glue of all eight moth-catching species. Further, we quantified the presence of a previously reported but unidentified compound that appears in the glue of all moth specialists. These silks can provide insight into the functions of bioadhesives and inform our own synthetic adhesives.

Keywords: Cyrtarachninae; NMR; aggregate glue; arachnology; bolas; spider silk.

Figure 1

Aggregate glue and water-soluble component residue. To produce these samples, capture threads were washed and the resulting solution was freeze-dried. In these photographs, the residue from moth-specialist species appears to be different from that of generalist species. The residue of moth specialists was fluffier and more loosely packed (left column). The generalist species had a more densely packed and crystalline residue (right column; see also [26]).

Figure 2

¹H solution-state 400 MHz NMR spectrum of water-soluble LMMCs dissolved in D₂O from the aggregate glue of C. akermani. The pie chart displays the relative abundance of each LMMC extracted from the aggregate glue. C. akermani had less chemical diversity than any other moth specialist.

Figure 3

¹H solution-state 400 MHz NMR spectrum of water-soluble LMMCs dissolved in D₂O from the aggregate glue of M. hutchinsoni. The pie chart displays the relative abundance of each LMMC extracted from the aggregate glue. M. hutchinsoni contained taurine, which was absent in the other bolas spider, C. akermani.

Figure 4

¹H solution-state 400 MHz NMR spectrum of water-soluble LMMCs dissolved in D₂O from the aggregate glue of P. walleri. The pie chart displays the relative abundance of each LMMC extracted from the aggregate glue. Due to peak overlap, we were not able to separately estimate NAT and NAP.

Figure 5

¹H solution-state 400 MHz NMR spectrum of water-soluble LMMCs dissolved in D₂O from the aggregate glue of C. ixoides. The pie chart displays the relative abundance of each LMMC extracted from the aggregate glue. Due to peak overlap, we were not able to separately estimate NAT and NAP.

Figure 6

¹H solution-state 400 MHz NMR spectrum of water-soluble LMMCs dissolved in D₂O from the aggregate glue of C. bufo. The pie chart displays the relative abundance of each LMMC extracted from the aggregate glue. Due to peak overlap, we were not able to separately estimate NAT and NAP.

Figure 7

¹H solution-state 400 MHz NMR spectrum of water-soluble LMMCs dissolved in D₂O from the aggregate glue of G. cancriformis. The chart displays the relative abundance of each LMMC extracted from the aggregate glue. G. cancriformis was the only species to contain proline and the only generalist to contain traces of Compound X.

Figure 8

Chemical structure of water-soluble compounds identified in spider-silk samples.

Figure 9

Aggregate glue LMMCs vary in proportion and presence. Moth specialists and G. cancriformis were the only species in which we detected Compound X (CMX; dark red). Bolas spiders C. akermani and M. hutchinsoni had larger proportions of CMX than other moth-catching taxa. Moth-specialist taxa were also characterized by a lack of GABamide (purple). The relative proportions of CMX and GABamide are highlighted in the column on the right. Overall, the glue of moth specialists was chemically distinct from that of generalist species. The phylogenetic topology is based on an ML tree created from an independent set of molecular characters (Figure S1). Some moth-specialist species had peak interference which did not allow us to separate the amount of NAT from NAP, so were estimated together (shown as a striped pattern).

Figure 10

Character evolution of the LMMCs. Moth-catching specialists (taxa names in bold) form a monophyletic taxon, sharing a hypothetical common ancestor (HCA), with its variable character states reconstructed as either present (filled blue circle) or absent (empty blue circle). At or after that HCA, the evolution of a particular character state in the specialists occurs in one of three patterns: (1) loss (ALA, NAT, NAP, and GAB; red minus signs), (2) gain (PKY, PKZ, and CMX; red plus signs), or (3) convergence (TAU). Ancestral character states are reconstructed using simple parsimony, with absence indicated as a solid white line and presence indicated as a solid blue line; the one equivocal state at the root of the NAP tree is a combination of blue and white lines. The character × taxon matrix shows the coding of the characters as absent (0) or present (1) and includes all 14 LMMCs.