χ-Conotoxins are an Evolutionary Innovation of Mollusk-Hunting Cone Snails as a Counter-Adaptation to Prey Defense

Abstract

Mollusk-hunting (molluscivorous) cone snails belong to a monophyletic group in Conus, a genus of venomous marine snails. The molluscivorous lineage evolved from ancestral worm-hunting (vermivorous) snails ∼18 Ma. To enable the shift to a molluscivorous lifestyle, molluscivorous cone snails must solve biological problems encountered when hunting other gastropods, namely: (i) preventing prey escape and (ii) overcoming the formidable defense of the prey in the form of the molluscan shell, a problem unique to molluscivorous Conus. Here, we show that χ-conotoxins, peptides exclusively expressed in the venoms of molluscivorous Conus, provide solutions to the above problems. Injecting χ-conotoxins into the gastropod mollusk Aplysia californica results in impaired locomotion and uncoordinated hyperactivity. Impaired locomotion impedes escape, and a hyperactive snail will likely emerge from its shell, negating the protection the shell provides. Thus, χ-conotoxins are an evolutionary innovation that accompanied the emergence of molluscivory in Conus and provide solutions to problems posed by hunting other snails.

Keywords: conotoxins; emergence of complex phenotypes; evolutionary innovation; mollusk-hunting cone snails; χ-conotoxin.

Fig. 1.

Reconstruction of Conus evolutionary history, highlighting the different feeding groups and divergence timing analysis. Multilocus-derived multispecies divergence timing analysis tree to display the taxa analyzed in this study indicating a single evolution of molluscivory. Feeding groups are highlighted: gray (vermivores), green (piscivores), and orange (molluscivores). Colored circles indicate estimated divergence times for nodes of interest (see supplementary fig. S4, Supplementary Material online for credibility intervals) obtained with BEAST using species coalescent topology generated with ASTRAL-III as input tree (see supplementary fig. S3, Supplementary Material online for ASTRAL tree). Values shown are in millions of years. Support values for all the nodes are found in supplementary fig. S3, Supplementary Material online (see also supplementary figs. S1 and S2, Supplementary Material online). The node corresponding to the ancestor of Tesseliconus and the mollusk-hunting species was recovered with maximum support. Outgroup refers to Conasprella species (see supplementary figs. S1 and S2, Supplementary Material online for additional information). Worm and fish-hunting Conus species represented in the picture are: (1) C. tribblei; (2) C. imperialis; (3) C. pertusus; (4) C. planorbis; (5) C. quercinus; (6) C. musicus; (7) C. geographus; (8) C. dusaveli; (9) C. striatus; (10) C. rolani; (11) C. lynceus; (12) C. tessulatus. Mollusk hunters are as follows: (I) C. cordigera; (II) C. bandanus; (III) C. marmoreus; (IV) C. ammiralis; (V) C. gloriamaris; (VI) C. textile; (VII) C. crocatus; (VIII) C. omaria; (IX) C. purus; (X) C. furvus; (XI) C. thalassiarchus.

Fig. 2.

χ-Conopeptide is highly expressed in the venom of C. aulicus and these peptides form a structurally and pharmacologically distinct family in the T-superfamily of conotoxins. a) HPLC chromatogram showing the absorbance profile at 220 nm of crude C. aulicus venom fractionated on a preparative C₁₈ column using a linear gradient of 1.3% B₉₀/min at a flow rate of 20 mL/min. The fraction marked by the arrow exhibited activity against hNET, as described in the Results section. b) Analytical HPLC chromatogram profile of the subfractionation of the active component in (a). The subfractionation was done using a linear gradient of 30% to 50% B₉₀ for 20 min at a flow rate of 1 mL/min. The active peak is indicated by the arrow. Note that the hNET active fraction is the major component of this solution. c) Analytical HPLC chromatogram profile showing the homogeneity of the active fraction in (b). The HPLC experiment was done using the same linear gradient described in (b). The component that inhibits hNET has the sequence: SVCCGYKLCFOC# (O = hydroxyproline, # = C-terminal amidation). This purified fraction is designated as χ-AuId. d) Dose-dependent inhibition of hNET by χ-AuId. IC₅₀ value of χ-AuId against hNET is 2.6 ± 0.4 μM. This value is the average from three experiments done in triplicate. e) Comparison of the cysteine framework between χ-conotoxins and canonical T-superfamily peptides. f) Sequence logo of χ-conotoxins. Conservative substitutions in amino acids between Cys 2 and Cys 3 are observed in these peptides. The sequences are derived from Mr1.1: C. marmoreus; Bn1.5: C. bandanus; Lg1.1: C. legatus; Tx1.6: C. textile; Af1.3: C. ammiralis; Au1.4: C. aulicus; Pur1.1: C. purus; Cc1.1: C. crocatus; and Fv1.5: C. furvus.

Fig. 3.

BLOSUM62 CLANS cluster map of χ-conotoxins and other T-superfamily toxins. Colored circles represent individual toxin precursor sequences, and the connecting edges show the blastp P-values above 1e−20 between the connected circles. The circles are color coded according to prey preferences (gray: vermivores, green: piscivores, orange: molluscivores). The χ-conotoxins (represented by a blue prism) are all part of a lineage of 2-loop Ts, which are exclusively found in molluscivores.

Fig. 4.

Behavioral phenotype after injecting χ-AuId into A. californica and images of C. bandanus envenomation progression. Video traces of the Aplysia body position showing the path traveled over a 15 min period after injection with: a) saline control, b) 0.01 nmol serotonin (5-HT), and c) 10 nmol χ-AuId. d) Quantification of the distance traveled (in number of pixels). The bar for each treatment (n = 3, open circles) represents the distance traveled over a 5 min period (see inset). The P-values are: χ-AuId (0 to 5 min, P = 0.0067; 5 to 10 min, P = 0.0459; 10 to 15 min, P = 5.7E-5), 5-HT (0 to 5 min, P = 0.006; 5 to 10 min, P = 0.02; 10 to 15 min, P = 0.084). Error bars represent standard deviation. e) to g) series of photographs showing events of C. bandanus envenomation of a prey snail. e) Conus bandanus envenomates prey. f) Prey tries to withdraw into the shell (note the operculum almost sealing off the shell). g) Minutes later, the prey emerges straight into the rostrum (false mouth) of C. bandanus.