Unraveling the ecological chemistry of prey capture by the Trichonephila clavipes and Trichonephila plumipes spider web through omics approaches

Author(s)

Franciele Grego Esteves

• Document type: Doctoral Thesis
• Press: Rio Claro. 2021-10-22.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Rio Claro
• Defense date: 2021-09-10
• Advisor: Mario Sergio Palma; José Roberto Aparecido dos Santos Pinto

Abstract

Trichonephila clavipes spider belongs to the group of orb-web weaving spiders, which build large and circular webs to capture their prey. Through aggregate silkproducing gland, these spiders deposit oily droplets that coat the threads of the capture spiral. These oily droplets contain many lipid vesicles that trap solutions of proteins, peptides, and low molecular weight compounds. Among these molecules, many are low molecular weight toxins and toxic proteins which can, for example, paralyze prey at the moment they come in contact with the web. It is further speculated that when an insect-prey is trapped by the web, droplets and lipid vesicles play an important role in penetrating, storing, transporting, and ultimately releasing the toxins into the prey's body. Considering this, using mass spectrometry analyses, it was possible to identify six classes of lipids that constitute the web droplets and vesicles lipids. Those classes include mainly fatty acids and esters, as well as glycerophospholipids and sphingolipids, and also glycerolipids and sterolipids. Furthermore, through the quantitative analysis of the web vesicular extract, it was possible to estimate that, for a single web, the vesicular extract presented an average of 14,709,800 lipid vesicles, with a value expressed in mass of 700µg of protein/peptides; so that each vesicle possibly traps inside ≅ 47 pg of protein/peptides, probably corresponds to the toxins that paralyze the insect-prey at the time they are released into the insect body. So far, not much is known about the peptide composition of this web. Thus, through the peptide profile analysis by LC-MS/MS, a total of 729 peptides were identified in the T. clavipes spider web silk and in its silk-producing glands. Thirteen peptides were selected and synthesized in solid phase, and their toxic potential could be evaluated in insect-toxicity assays. It was observed that both the web vesicular extract and the peptides named Trichonephiline-1, -2, -3, -4, -8, 10 and 13 showed toxic effects when injected into the bee hemocele. Web vesicular extract showed LD50 value = 0.323 ng/mg while Trichonephiline-1, -2, -3 peptides showed LD50 value = 1.06 ng/mg, 0.846 ng/mg and 1.06 ng/mg, respectively. Furthermore, when subjected to Fluorescence Imaging Plate Reader (FLIPR) assays, Trichonephiline-1, -2, -3, -4, -8, 10 and 13 peptides also showed inhibitory action on sodium (Nav) and calcium (Cav2.2) ion channels. The Trichonephiline-8 peptide also inhibited calcium ion channels (Cav1.3) and the nicotinic acetylcholine receptor alpha 7 (nAChRα7), while the Trichonephiline10 peptide inhibited only the nAChRα7 receptor. In addition to potential insecticidal peptides involved in prey capture, the peptide Trichonephiline-6 was identified with antibiotic action (MIC <= 0.25 ug/mL) for Gram+ Staphylococcus aureus bacteria. The total web extract also showed antibiotic action with MIC value ≤ 2 µg/mL for GramPseudomonas aeruginosa bacteria. Both web silk and Trichonephiline-6 peptide may be contributing to the defense mechanism of the web, or even the developing spider eggs, resist microbial decay. Finally, chemical imaging analysis by mass spectrometry (IMS-MALDI) of T. plumipes spider web was performed for the first time, which showed that the distribution of peptides in the web occurs heterogeneously. Thus, this chapter provides a better chemical-ecological understanding of prey capture by the webs of the genus Trichonephila spiders, and serves as inspiration for the development of new insecticide-selectives and/or antibiotics. (AU)

• FAPESP's process: 17/10373-0 - Profiling the peptidomic and structural-functional characterization of lipid vesicles present in the Nephila clavipes web spider
• Grantee: Franciele Grego Esteves
• Support Opportunities: Scholarships in Brazil - Doctorate