No abstract

Spiders have distinct predatory behaviours selected along Araneae’s evolutionary history but are mainly based on the use of venom for prey paralysis. Uloboridae spiders have lost their venom glands secondarily during evolution. Because of this, they immobilise their prey by extensively wrapping, and digestion starts with the addition of digestive fluid. During the extra-oral digestion, the digestive fluid liquefies both the prey and the AcSp2 spidroins from the web fibres. Despite the efficiency of this process, the cocktail of enzymes involved in digestion in Uloboridae spiders remains unknown. In this study, the protein content in the midgut of Uloborus sp. was evaluated through enzymatic, proteomic, and phylogenetic analysis. Hydrolases such as peptidases (endo and exopeptidases: cysteine, serine, and metallopeptidases), carbohydrases (alpha-amylase, chitinase, and alpha-mannosidase), and lipases were biochemically assayed, and 50 proteins (annotated as enzymes, structural proteins, and toxins) were identified, evidencing the identity between the digestive enzymes present in venomous and non-venomous spiders. Even enzymes thought to be unique to venom, including enzymes such as sphingomyelinase D, were found in the digestive system of non-venomous spiders, suggesting a common origin between digestive enzymes and enzymes present in venoms. This is the first characterization of the molecules involved in the digestive process and the midgut protein content of a non-venomous spider.

Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.