Data from: High throughput techniques to reveal the molecular physiology and evolution of digestion in spiders

Background: Spiders are known for their predatory efficiency and for their high capacity of digesting relatively large prey. They do this by combining both extracorporeal and intracellular digestion. Whereas many high throughput (“-omics”) techniques focus on biomolecules in spider venom, so far this approach has not yet been applied to investigate the protein composition of spider midgut diverticula (MD) and digestive fluid (DF). Results: We here report on our investigations of both MD and DF of the spider Nephilingis (Nephilengys) cruentata through the use of next generation sequencing and shotgun proteomics. This shows that the DF is composed of a variety of hydrolases including peptidases, carbohydrases, lipases and nuclease, as well as of toxins and regulatory proteins. We detect 25 astacins in the DF. Phylogenetic analysis of the corresponding transcript(s) in Arachnida suggests that astacins have acquired an unprecedented role for extracorporeal digestion in Araneae, with different orthologs used by each family. The results of a comparative study of spiders in distinct physiological conditions allow us to propose some digestion mechanisms in this interesting animal taxon. Conclusion: All the high throughput data allowed the demonstration that DF is a secretion originating from the MD. We identified enzymes involved in the extracellular and intracellular phases of digestion. Besides that, data analyses show a large gene duplication event in Araneae digestive process evolution, mainly of astacin genes. We were also able to identify proteins expressed and translated in the digestive system, which until now had been exclusively associated to venom glands

High throughput techniques to reveal the molecular physiology and evolution of digestion in spiders

BACKGROUND: Spiders are known for their predatory efficiency and for their high capacity of digesting relatively large prey. They do this by combining both extracorporeal and intracellular digestion. Whereas many high throughput (“-omics”) techniques focus on biomolecules in spider venom, so far this approach has not yet been applied to investigate the protein composition of spider midgut diverticula (MD) and digestive fluid (DF). RESULTS: We here report on our investigations of both MD and DF of the spider Nephilingis (Nephilengys) cruentata through the use of next generation sequencing and shotgun proteomics. This shows that the DF is composed of a variety of hydrolases including peptidases, carbohydrases, lipases and nuclease, as well as of toxins and regulatory proteins. We detect 25 astacins in the DF. Phylogenetic analysis of the corresponding transcript(s) in Arachnida suggests that astacins have acquired an unprecedented role for extracorporeal digestion in Araneae, with different orthologs used by each family. The results of a comparative study of spiders in distinct physiological conditions allow us to propose some digestion mechanisms in this interesting animal taxon. CONCLUSION: All the high throughput data allowed the demonstration that DF is a secretion originating from the MD. We identified enzymes involved in the extracellular and intracellular phases of digestion. Besides that, data analyses show a large gene duplication event in Araneae digestive process evolution, mainly of astacin genes. We were also able to identify proteins expressed and translated in the digestive system, which until now had been exclusively associated to venom glands. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-3048-9) contains supplementary material, which is available to authorized users

Strahlenschutz-Praxis : Zeitschrift für den sicheren Umgang mit ionisierender und nichtionisierender Strahlung ; Organ des Fachverbandes für Strahlenschutz e.V.

Scorpions are among the oldest terrestrial arthropods and they have passed through small morphological changes during their evolutionary history on land. They are efficient predators capable of capturing and consuming large preys and due to envenomation these animals can become a human health challenge. Understanding the physiology of scorpions can not only lead to evolutionary insights but also is a crucial step in the development of control strategies. However, the digestive process in scorpions has been scarcely studied. In this work, we describe the combinatory use of next generation sequencing, proteomic analysis and biochemical assays in order to investigate the digestive process in the yellow scorpion Tityus serrulatus, mainly focusing in the initial protein digestion. The transcriptome generated database allowed the quantitative identification by mass spectrometry of different enzymes and proteins involved in digestion. All the results suggested that cysteine cathepsins play an important role in protein digestion. Two digestive cysteine cathepsins were isolated and characterized presenting acidic characteristics (pH optima and stability), zymogen conversion to the mature form after acidic activation and a cross-class inhibition by pepstatin. A more elucidative picture of the molecular mechanism of digestion in a scorpion was proposed based on our results from Tityus serrulatus. The midgut and midgut glands (MMG) are composed by secretory and digestive cells. In fasting animals, the secretory granules are ready for the next predation event, containing enzymes needed for alkaline extra-oral digestion which will compose the digestive fluid, such as trypsins, astacins and chitinase. The digestive vacuoles are filled with an acidic proteolytic cocktail to the intracellular digestion composed by cathepsins L, B, F, D and legumain. Other proteins as lipases, carbohydrases, ctenitoxins and a chitolectin with a perithrophin domain were also detected. Evolutionarily, a large gene duplication of cathepsin L occurred in Arachnida with the sequences from ticks being completely divergent from other arachnids probably due to the particular selective pressures over this group

Phylogenetic relationships among endopeptidases present in MMG of the scorpion <i>Tityus serrulatus</i> using maximum likelihood algorithm.

<p>Sequences displayed in red were identified by proteomics.</p

Hydrophobic chromatographic fractioning of <i>Tityus serrulatus</i> MMG homogenate.

<p>MMG homogenate was fractioned with 50% ammonium sulfate on a HiTrap Butyl column (GE) equilibrated in 50 mM phosphate buffer (pH 6.0). Elution was performed using a gradient of 1.7–0 M ammonium sulfate in the same buffer. (A) Activity of each fraction against 10 μM Z-FR-MCA was measured in 100 mM Tris-HCl buffer (pH 8.0) containing 10 mM CaCl₂ (●) or in the presence of 5.0 mM benzamidine (○). (B) The activity of each fraction against 10 μM Z-FR-MCA was measured in 100 mM CP-buffer (pH 5.5) containing 3.0 mM cysteine and 3.0 mM EDTA in the absence (●) and presence of different peptidase inhibitors: (□) 10 μM E-64; (○) 1.0 mM PMSF; (Δ) 10 μM pepstatin.</p

Possible digestive enzymes identified after the transcriptomic analysis in the midgut and midgut glands of the scorpion <i>Tityus serrulatus</i>.

<p>Possible digestive enzymes identified after the transcriptomic analysis in the midgut and midgut glands of the scorpion <i>Tityus serrulatus</i>.</p

Quantitative analysis of selected proteins likely involved in digestion.

<p>Values are means and SEM from relative normalized spectra counting (NSC) calculated based on possible digestive enzymes identified. Shotgun proteomics experiment on triplicate samples with MMG of fasting and fed scorpions.</p

Acid activation of cysteine endopeptidases from <i>Tityus serrulatus´</i> MMG.

<p>Effect of incubating MMG homogenate (A) at 30°C for 60 minutes under different pH conditions. (B) Effect of time on acidic activation of cysteine peptidases from <i>Tityus serrulatus</i> MMG homogenate. After incubation in acidic buffer (pH 2.6), 2 μl of each enzyme preparation was assayed in 200 μl of 0.1 M CP buffer (pH 5.5) with Z-FR-MCA to measure activity at constant pH. Activity increase was calculated as ratio of incubated enzyme activity over non-incubated control activity. All buffers used for activation (0.1 M CP, pH 2.6–7.0) and activity assays contained 3.0 mM cysteine and 3.0 mM EDTA.</p

General morphology of scorpion digestive system and its location.

<p>Schematic ventral (A) and lateral (B) views of scorpion digestive system and its divisions. Right picture (A) represents ventral view of <i>Tityus serrulatus</i> MMG. PMG, prosomal midgut. Fig 1B was modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123841#pone.0123841.ref002" target="_blank">2</a>].</p

Peptidase absolute and specific activities involved in protein digestion in MMG from the scorpion <i>Tityus serrulatus</i> using different substrates.

<p>Peptidase absolute and specific activities involved in protein digestion in MMG from the scorpion <i>Tityus serrulatus</i> using different substrates.</p