Web Toxinomics: A New Focus For The Orb-Web-Spiders

Toxinology Research

Palma, M.S¹; Salles, H.C.; Marques, M.R.¹; Mendes, M.A¹.; Souza, B.M¹.; Cesar, L.M.M.; Volsi, E.C.F.R¹.; Tormena, C. F².; Rittner, R³.

1 Dept. Biology-CEIS / IBRC - UNESP - Rio Claro, SP, CEP: 13506-900 - Brazil; 2 Dept. Chemistry, F.F.C.L.R.P- São Paulo University, Ribeirão Preto, SP, Brazil; 3 Physical Organic Chemistry Laboratory- Chemistry Institute- UNICAMP - Campinas - CEP: 13083-970 – Brazil

The Nephilinae spiders are sedentary animals, which expend the most their lives around orbital webs, which are used for nesting, reproduction and for prey capture. Usually viscous droplets cover the capture threads of their webs as part of the strategy for prey paralysis/killing; small / medium sized Arthropods captured by the webs, generally show toxicity signs even prior venom injection by the spiders. When these viscous (oily) droplets are extracted from the webs and used in insect-toxicity assays, it becomes evident that the content of the viscous droplets are at least so toxic as the spider venom itself. By using transmittance electronic microscopy and scanning electronic microscopy we demonstrated that the viscous droplets of the capture treads present a complex structure, constituted of hundreds of small lipid vesicles, which disrupt by contact with the bodies of prey Arthropods. Cytochemical analysis revealed that these small vesicles are filled either with aqueous solutions containing mainly proteins/peptides or filled with heterogeneous lipid material.

In order to identify the toxic components from the web of Nephilinae spiders, the webs of Nephilaclavipes and Nephilengyscruentata were collected in the nature, separated into three solid aliquots, which were individually washed either with hexane or 10 mM ammonium bicarbonate pH 6.8 or 50% (v/v) MeCN. The fraction hexane soluble was directly analyzed by GC-MS, revealing the presence of a series of fatty acids, which apparently seem to be used as basic matrix both for the viscous droplets and for the small vesicles. However, a toxic leukotriene analog was also identified.

The web extract soluble in 50%(v/v) MeCN was fractionated by using RP-HPLC in a C-18 semi preparative column (SHISEIDO 10 x 250 mm); elution was carried out in a linear gradient concentration from 5% to 60% (v/v) MeCN (containing 0.1% TFA). The eluting fractions were collected and assayed for insect toxicity by using honeybees as model insects; the active fractions were analyzed by ESI-MS, ESI-MS/MS, H¹-NMR, C¹³-NMR and when necessary ICP spectroscopy was also used. This experimental approach revealed the presence of at least three novel types of very potent insecticide toxins: tetrahydrobetacarbolines, iron phenyl hydrazil ethyl alcohol and hydroxymethyl aminomethyl-N-carbonylamino methylpropiophenone.

The web extract soluble in 10 mMammonium bicarbonate pH 6.8 revealed to be rich in proteins and peptides and was submitted to a shot-gun proteomic analysis in order to identify the polypeptide components of this extract. The proteins identified were: ferrichrometransport system permease, chain A of the exfoliativeToxin A, an ATP-dependent protein transporter and a calcium binding protein. Toxins of at least three different chemical natures were identified in the webs of the Nephilinaespiders, which apparently seem to act synergistically to cause prey paralysis/death.

The wide arsenal of toxins, presenting novel chemical structures and interesting mechanisms of action, certainly will enlarge the possibilities to implement innovative researches in orb-web-spiders toxinology in a forthcoming future, and even be used as source of bioprospection for the development of novel pharmaceuticals/agrochemicals.

Supported by: CAT/CEPID/FAPESP; CNPq