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. 2016 Sep 7;17(1):716.
doi: 10.1186/s12864-016-3048-9.

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

Felipe J Fuzita  1   2 , Martijn W H Pinkse  3 , José S L Patane  4 , Peter D E M Verhaert  3   5 , Adriana R Lopes  6
Affiliations

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

Felipe J Fuzita et al. BMC Genomics. .

Abstract

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.

Keywords: Arachnida; Astacin; Digestion; Enzyme; High throughput (-omics) techniques; Nephilingis (Nephilengys cruentata); Spider.

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Figures

Fig. 1
Fig. 1
Venn diagram of proteome data of digestive fluid (DF) and opisthosomal midgut diverticula (MD) samples of N. cruentata. a All identified proteins in DF and MD from fasting and 9 h fed spiders. b Digestive enzymes identified in DF and MD samples. All DF samples obtained from fasting and different periods of feeding (1, 3, 9, 25 and 48 h) were used
Fig. 2
Fig. 2
Sum of all distinct elected digestive enzyme types and respective number of isoforms identified by shotgun proteomics in MD from fed and fast animals and DF
Fig. 3
Fig. 3
Proteome quantification using normalized spectral abundance factor (NSAF). Values represent mean percentages of digestive enzymes using 3 distinct biological replicates for midgut diverticula samples. For comparison, percentages were calculated relative only to digestive enzymes from Table 1. In DF 17 different samples were analyzed all together. a Fed. b Fasting. c DF. In fasting sample analysis only carbohydrases and exopeptidases with NSAF values equal or greater than 0.5 % are shown (for optimal visualization)
Fig. 4
Fig. 4
Phylogenetic analysis of arachnid astacin DNA sequences using Maximum Likelihood algorithm. UFBoot values (1000 pseudoreplicates) are shown for each node. Colored proteins in red or blue were identified by mass spectrometry in the present work or by other authors [5, 16], respectively. Letters after sequence names indicate tissue location by mass spectrometry, B, whole body; V, venom; S, silk glands; D, digestive fluid; M, midgut diverticula; O, opisthosoma; P, prosoma; H, hemolymph. Sequences were named with a number after an abbreviation used for each species as follows: Smimo, Stegodyphus mimosarum; Ptepi, Parasteatoda tepidariorum; Ncrue, Nephilingis cruentata; Ageni, Acanthoscurria geniculata; Lhesp; Latrodectus hesperus; Tserr, Tityus serrulatus; Mmart, Mesobuthus martensii; Iscap, Ixodes scapularis; Irici, Ixodes ricinus; Amacu, Amblyomma maculatum; Turti, Tetranychus urticae; Lpoly, Limulus polyphemus; Aast, Astacus astacus; Lvann, Litopenaeus vannamei; Pcamt, Paralithodes camtschaticus. Additional file 11 shows the accession number or contig IDs to all sequences used
Fig. 5
Fig. 5
Reconciliation of ML gene tree with species tree according to [17]. Red and green numbers in each branch respectively represents duplication and losses
Fig. 6
Fig. 6
Schematic representation of digestive and secretory cells present in MD of N. cruentata. The content of the secretory vesicles present in secretory cells represents the DF. F: partially digested food; PhA2: phospholipase A2
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