Digestive enzymes and sphingomyelinase D in spiders without venom (Uloboridae)

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.

Figure 1

SDS PAGE profile of midgut samples from Uloborus sp. About 10 μg of proteins from the midgut of Uloborus sp. was submitted to electrophoresis in a 12% polyacrylamide gel at 150 V. MWS—Molecular Weight Standard, 97 kDa—Phosphorylase B, 66 kDa—Albumin, 45 kDa—Albumin from egg, 30 kDa—Carbonic anhydrase, 20.1 kDa—Trypsin inhibitor and 14.4 kDa—Lysozyme. The gel was silver stained according to.

Figure 2

Values of endopeptidases, exopeptidases, carbohydrases and lipase specific activities from the midgut diverticula (MD) of Uloborus sp. Enzyme assays were performed as shown in Table 1. N = 5 to endopeptidases (2A); N = 6 to exopeptidases (2B); N = 8 to carbohydrases (2C), except to alpha-amylase (N = 4) and N = 8 to lipase (2D). Colored bars represent specific activity, mean and ± standard deviation of biological replicates for a specific enzyme according to color legend. * Means no activity detected.

Figure 3

The amount and protein categories by Uloborus sp. midgut proteome. Proteins were set apart into six different categories and their amount in Uloborus sp. midgut proteome: structural (7), carbohydrases (3), peptidases (21), phospholipases (1), toxins (7) and metabolism (11), summing a total of 50 proteins. Plot was generated in Excel.

Figure 4

Spider venom and midgut SMaseD multiple sequence alignment and their classification by cysteine conservation. SMaseD of class I, class IIa and class IIb from L. venom gland were aligned against SMaseD from abdomen A. geniculata, midgut N. cruentata, digestive fluid S. mimosarum, and whole body S. dumicola and O. yesoensis in multiple sequence alignment (MUSCLE software), to classify the SMaseD found in the midgut/digestive fluid of spiders. Sequences with only one disulfide bond between Cys97 and Cys103 are classified as “Class I SMaseD” and sequences with an additional disulfide bond between Cys99 and Cys 250 are classified as “Class II SMaseD''. Red box: 1º disulfide bond cysteine conservation; Purple box: 2º disulfide bond cysteine conservation; Blue line/arrow; columns of amino acids positions hidden (95–108 and 240–253). Midgut/digestive fluid SMaseD sequences only conserve the four cysteine residues (Cys97, Cys99, Cys 103, and Cys 250). Alignment was generated in Jalview (https://www.jalview.org).

Figure 5

Spider venom and midgut SMaseD multiple sequence alignment, their classification by glycine and proline substitutions, and conservation of residues involved in catalysis. SMaseD of class I, class IIa and class IIb from L. venom gland were aligned against SMaseD from abdomen A. geniculata, midgut N. cruentata, digestive fluid S. mimosarum, and whole body S. dumicola and O. yesoensis in multiple sequence alignment (MUSCLE software) , to classify the SMaseD found in midgut/digestive fluid of spiders. Most of the class I and class IIa SMaseD conserve the glycine and proline (purple highlighted and red arrowed indicated) residues, and class IIb SMaseD has a substitution in glycine/proline residues. As well, the conservation of histidines (His57 and His93), glutamic acid (Glu77), aspartic acid (Asp79), lysine (Lys139), tyrosine (Tyr278), and tryptophan (Trp280), involved in ion magnesium coordination and substrate recognition. Purple box: conserved histidine, glutamic acid, aspartic acid, lysine, glycine, and proline; Green box: conserved tyrosine; Blue box: conserved tryptophan. Midgut/digestive fluid SMaseD sequences only substitute the glycine residue (Gly141). Alignment was generated in Jalview (https://www.jalview.org.

Figure 6

Maximum Likelihood phylogenetic tree from SMaseD sequences. A Maximum Likelihood phylogenetic tree analysis by IQ Tree software and coloured by ITOL (https://itol.embl.de), composed of midgut, venom gland sequences from spiders. Bootstrap test of phylogeny was applied with 1000 resampling. All sequences are listed in Supplementary Table S5.

Figure 7

Female of Uloborus sp. feeding on a prey item captured in the field (picture from Marcelo de Oliveira Gonzaga).