Male tarsi specific odorant-binding proteins in the diving beetle Cybister japonicus sharp

Abstract

Odorant binding proteins (OBPs) play critical roles in chemical communication of insects, as they recognize and transport environmental chemical signals to receptors. The diving beetle Cybister japonicus Sharp shows a remarkable sexual dimorphism. The foreleg tarsi of males are equipped with large suction cups, believed to help holding the female during underwater courtship and mating. Here, we identified two OBPs highly and specifically expressed in male tarsi, suggesting important functions of these structures in chemical communication. The first protein, CjapOBP1, exhibits the 6 conserved cysteines motif of classic OBPs, while the second, CjapOBP2, contains only four cysteines and can be assigned to the sub-class of C-minus OBPs. Both proteins were expressed in a bacterial system and the purified recombinant proteins were used to for antibodies preparation. Western Blot analysis showed that CjapOBP1 is predominantly expressed in male tarsi and could be also detected in antennae and palpi of both sexes, while CjapOBP2, besides male tarsi, is also present in testis. Ligand-binding experiments showed a good binding affinity between CjapOBP1, CjapOBP2 and citral and coniferyl aldehyde, respectively. These results support a possible function of these two OBPs in the male foreleg tarsi of diving beetles in chemical communication.

Figure 1. Morphology of proleg tarsi of C. japonicus.

The proleg of male (A) and female (B) are sexual dimorphism, in that the prolegs of males are equipped with large suction cups (C) absented in females, and bear specialized adhesive setae on their ventral side. Each spatula seta, labeled with the rectangle, is shown at higher magnification in (D), and connects to the palette with an off-centre stalk and its ventral surface has an oval-shaped sucker. Scale bar: 1 mm for (C), and 30 µm for (D).

Figure 2. Expression of CjapOBP1 and CjapOBP2 from the tarsi of male C. japonicus.

(A) SDS-PAGE analysis of tatsi from both male and female showed the abundant presence of low molecular weight bands mainly in the crude extracts from the tarsi of male C. japonicus. (B) A crude extract of male tarsi was separated on a native gel, and two fast migrating bands were selected for N-terminal sequencing. mT, male tarsi; fT, female tarsi. Molecular mass markers (M) are (from the top): 97 kDa (phosphorylase B), 66 kDa (BSA), 45 kDa (ovalbumin), 29 kDa (carbonic anhydrase), 20 kDa (trypsin inhibitor) and 14 kDa (α-lactalbumin).

Figure 3. Structure of CjapOBPs and phylogenetic relationship of CjapOBPs with other beetle OBPs.

(A) The newly identified CjapOBP1 (a typical OBP with six cysteines) and CjapOBP2 (a C-minus OBP with only four cysteines) are aligned with seven OBP proteins from other Coleoptera. The asterisks indicate the conserved amino acids. The conserved cysteine residues are highlighted in blue box. The phylogenic tree (B) was constructed with amino acid sequences of CjapOBP1 and CjapOBP2 as well as seven OBP proteins from other beetles using MEGA 6 software. The tertiary structures of CjapOBP1 (C) and CjapOBP2 (D) were stimulated based on the selected protein templates from I-TASSER.

Figure 4. SDS-PAGE and Western blot of crude extracts from different parts of the body of adult C.japonicus.

(A) SDS-PAGE analysis of crude extracts from different tissues of male and female C. japonicas, showed the presence of low molecular weight bands mainly in the male tarsi sample. Western blot membrane after reaction with polyclonal antiserum against CjapOBP1 (B) or CjapOBP2 (C) and peroxidase-conjugated second antibody. Ant, antennae; Tar, tarsi; Pal, palpi; AG, accessory gland; and Tes, testis. Molecular mass markers (M) are (from the top): 97 kDa (phosphorylase B), 66 kDa (BSA), 45 kDa (ovalbumin), 29 kDa (carbonic anhydrase), 20 kDa (trypsin inhibitor) and 14 kDa (α-lactalbumin).

Figure 5. Affinity of CjapOBPs to candidate chemical ligands.

(A) Both CjapOBP1 and CjapOBP2 bind the fluorescent probe 1-NPN, with dissociation constants of 3.3 μM and 2.1 μM, respectively. (B) Competitive binding curves of selected ligands to the two CjapOBPs. Mixtures of proteins (5 μM) and 1-NPN (2 μM) were titrated with 1mM solutions of the ligands in methanol. Among the ligands tested, CjapOBP1 shows good affinity only to citral, and CjapOBP2 bind coniferyl aldehyde strongly, but not dihydroferulic acid, eugenol and α-methoxycinnamaldehyde, which are structurally related to coniferyl aldehyde. (C) The chemical structures of the chemicals applied.