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

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.

The diving beetle Cybister japonicus Sharp (Coleoptera: Dytiscidae) lives in stagnant waters 48,49 and is part of an important group of predatory insects. The beetles of the Dytiscidae family often show a range of peculiar secondary sexual characters in both males and females. The three basal segments of the pro-tarsi in males are usually equipped with various combinations of small and large suction cups. These cups are suggested to play roles in climbing, swimming and catching preys, as well as reproduction [50][51][52] . In such context, males use specialized proleg tarsi to adhere the slippery females elytra 50 .
In this study, we reported the first two OBPs in the diving beetle C. japonicas. The structure modeling, specialized expression in male tarsi and ligand-binding properties of these OBPs suggested their potential function in chemical communication between male and female diving beetles.

Results
Aim of the present work is to elucidate the function of the giant male front tarsi in water beetles through a study of the proteins expressed, focusing on odorant-binding proteins. The clear sexual dimorphism and the use of these anatomical structures in courtship and mating suggested that they might be involved in chemical communication.
Morphology of proleg tarsi of C. japonicus. Although the peculiar structure of male tarsi in water beetles has been reported 52 , we first undertook a morphological investigation to describe in detail such organ in the diving beetle C. japonicus Sharp using scanning electron microscopy (SEM). Figure 1 clearly evidences the remarkable sexual dimorphism of the tarsi. The prolegs of males are equipped with large suction cups, which are absent in females (Fig. 1A,B). The male proleg presents an expanded palette composed of protarsomeres bearing specialized adhesive setae on their ventral side (Fig. 1A). Each spatula seta connects to the palette with an off-centre stalk and its ventral surface has an oval-shaped sucker from which parallel channels extend distally (Fig. 1C,D). The structural elements of this organ are very similar to those reported for the male diving beetle Cybister rugosus 50 and proposed to control female movement.
Identification of odorant-binding proteins. We hypothesized that the exaggerated growth of male tarsi could be related to semiochemical delivery. Therefore we decided to investigate the protein composition of the tarsi in a comparative way between male and female diving beetles. Electrophoretic analysis (SDS-PAGE) of crude extracts from the tarsi of male and female C. japonicus showed the presence of low molecular weight bands mainly in the male sample ( Fig. 2A). Given the unusual abundance of these bands, migrating with apparent molecular masses compatible with the values expected for OBPs, we decided to investigate their protein composition. Thus, we separated a crude extract of male tarsi on a native gel (Fig. 2B) and selected two fast 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 ovalshaped sucker. Scale bar: 1 mm for (C), and 30 µm for (D).
Scientific RepoRts | 6:31848 | DOI: 10.1038/srep31848 migrating bands for N-terminal sequencing. Previous work had shown that generally OBPs migrate on the front of native gels and can be easily separated from other proteins 53 . The two selected bands were blotted onto PVDF membrane and subjected to N-terminal sequencing. We obtained the N-terminal 20 amino acids sequences LDDAQKAKFKAHYDLCVTET and ISPEQKEKMKKLHDECLHET for these two proteins and temporary named them as protein 1 and protein 2. Molecular Cloning, sequence and analysis and structural modeling of CjapOBPs. The information on the first 20 amino acids enabled us to design degenerate primers (Supplementary Table S1) at the 5′ ends of two genes. These degenerated primers together with oligo-(dT) 18 V at the 3′ end were used to amplify the relative genes with PCR. 5′ -RACE was then performed to complete the sequences at the 5′ end, so we obtained the full-length genes, as reported in the Materials and Methods section.
On the basis of their significant similarities with OBPs from other species in National Center for Biotechnology Information (NCBI) database, the two deduced protein sequences were named CjapOBP1 and CjapOBP2 and deposited in the GenBank database with accession numbers KX078770 and KX078771, respectively.
CjapOBP1 is a single polypeptide of 138 amino acids, with a calculated molecular weight for the mature protein of 14.1 kDa and a predicted isoelectric point of 5.49. CjapOBP2 is similar in length, with 132 amino acids, a calculated molecular weight of 13.6 kDa and an isoelectric point of 6.74 for the mature protein. While CjapOBP1 presents the six-cysteine signature of classic insect OBPs, CjapOBP2 contains only four cysteines and can be assigned to the family of C-minus OBPs. The sequences of the two OBPs of C. japonicus were aligned with seven representative OBPs from other Coleoptera species (Fig. 3A). Phylogenetic analysis was performed based on amino acid sequences alignment of these beetle OBPs. The phylogenetic tree was generated by MEGA 6.06 with the neighbor-joining algorithm. The nine beetle OBPs separated into two groups, one group with six cysteine residues and the other having four cysteine residues (Fig. 3B). The overall structures of CjapOBP1 and 2 (predicted by I-TASSER with highest ranking templates being 3qme and 3d77, respectively) consist of six helices (labelled α 1 to α 6) surrounding the hydrophobic ligand binding pocket (Fig. 3C,D). The predicted ligand binding site residues for CjapOBP1 include seven hydrophobic amino acids (Phe67, Phe72, Ile73, Ile79, Met84, Tyr119 and Tyr122) (Fig. 3C). The putative ligand binding site residues for CjapOBP2 consist of six hydrophobic amino acids (Met66, Phe67, Leu70, Phe72, Ile131 and Val132) (Fig. 3D).
Specific localization of CjapOBPs in male proleg tarsi. Using pET28a as a vector, we obtained the proteins fused with a His-tag fragment in high yields and in their soluble forms. Purification was performed by affinity chromatography on Ni-NTA His-Bind column. The electrophoresis analyses relative to expression and purification steps were reported in Supplementary Figure S1.
Western blot experiments, using a polyclonal antiserum raised against the purified protein, revealed that CjapOBP1 is very abundant in male tarsi, with relatively low expression in palpi and antennae of both sexes (Fig. 4). CjapOBP2, instead was detected in testis and tarsi of males (Fig. 4). Both proteins, therefore appear to be mainly expressed in the tarsi with a strong sex bias, suggesting their potential functions in chemical communication between male and female diving beetles.

Fluorescence binding assays.
To characterize the ligand-binding properties of the two OBPs, we performed fluorescence binding assays with several chemicals having different structures using N-phenyl-1-naphthylamine (1-NPN) as a fluorescent reporter. In the absence of any information about potential semiochemicals for this insect, as well as for related species, we selected some common plant volatiles, such as citral, coniferyl aldehyde, eugenol and 2-isobutyl-3-mthoxypyrazine, and structurally similar compounds. 1-NPN binds CjapOBP1 and CjapOBP2 with dissociation constants of 3.3 and 2.1 µ M respectively (Fig. 5A). Among the ligands tested, we identified a single good ligand for each protein, citral for CjapOBP1 and coniferyl aldehyde for CjapOBP2 (Fig. 5B,C). In particular, we can observe that several compounds structurally related to coniferyl aldehyde, such as dihydroferulic acid, eugenol and α -methoxycinnamaldehyde, did not show appreciable binding to CjapOBP2, indicating that this protein might have a rather narrow binding structure spectrum.

Discussion
In this study, we have identified two OBPs predominantly expressed in the frontal tarsi of male C. japonicus. The deduced amino acid sequences suggested that CjapOBP1 consists of a typical framework of OBPs (six-conserved cysteines), while CjapOBP2 contains only four cysteines and belongs to the family of C-minus OBPs (Fig. 3). Both proteins present high sequence similarities with OBPs of other Coleoptera. The first Coleoptera insect OBPs were identified from scarab beetles, specifically expressed in antennae, and associated with the pheromone recognition based on ligand-binding properties and single sensillum recordings evidences 42 . With the advantage of next-generation sequencing, a growing number of OBPs have also been reported in other Coleoptera species [54][55][56][57][58] . The diversified functions of these OBPs might be revealed in the future with future exploration.
C.japonicus has marked sexual dimorphism of the tarsi with SEM investigation (Fig. 1). It has been known that males use their giant front tarsi to hold on the back of females during their courtship [50][51][52] . The high expression of OBPs in the specialized front tarsi of males may suggest significant roles interfering with sex communication. Because during the underwater courtship the female C. japonicus could release sex pheromones used by males for partner localization that was observed in the diving beetle Rhantus suturalis 59 . Therefore it is reasonable to hypothesize that these OBPs specific expressed in male foreleg tarsi C. japonicus may be involved in the sex pheromone reception. Work is in progress in our lab to determine the functions of CjapOBP1 and CjapOBP2 through functional RNA interference and behavior studies. In swallowtail bufferfly Papilio xuthus, several chemosensory proteins and OBPs were identified in the female foreleg tarsi, which is served as a chemosensory organ in this species, suggesting an important function of these proteins in oviposition behavior of P. xuthus 60 . Most recently, two putative OBPs show abundant expression in palps and foreleg tarsi of Amblyomma americanum, which contain the olfactory Haller's organ. These A. americanum OBPs may also play roles in sex pheromones reception 61 . Future functional studies of these foreleg tarsi abundant OBPs will shed new lights on the understanding of molecular basis and evolution of chemoreception.
We can speculate that such proteins, rather than being involved in chemosensing, act as solubilisers and carriers of pheromones, and OBPs are transferred to females during such process together with their load of pheromones, as reported in several other species 12 .
Investigating the binding affinity and distribution of expression of OBPs is useful for understanding the physiological function and mechanism of olfactory recognition 37,62 . The fluorescent binding experiments showed that each protein is coupled with a single good ligand, citral for CjapOBP1 and coniferyl for CjapOBP2. The components of the sex pheromones in these beetles are unknown, but we can hypothesize that molecules structurally similar to citral and coniferyl aldehyde might represent good candidates, on the basis of our preliminary binding experiments.  Identification of C. japonicus OBPs. The different parts of the C.japonicus including antennae (30 pairs), tarsi (10 for male, and 30 for female), palpi (30 pairs), and sex organs (3) of both sexes were collected on ice and ground under liquid nitrogen. The samples were extracted with 0.3-1.0 mL 50 mM PBS (pH: 7.4), containing 1% PMSF and centrifuged at 13000 rpm for 20 min. Supernatants were collected and analyzed by 15% SDS-PAGE. For sequencing analysis, a sample of male tarsi was separated on a 15% native gel and blotted onto PVDF membrane. Fast migrating bands were subjected to automated Edman degradation, using a Procise 492 protein sequencer (Applied Biosystems, Foster City, Calif.), equipped with a 140C microgradient apparatus and a 785A UV detector (Applied Biosystems, Foster City, Calif.) for the automated identification of phenylthiohydantoin-amino acids.
RNA extraction, cDNA synthesis, and cloning. Total RNA was extracted using the TRIzol ® Reagent (Sigma, USA), following the manufacturer's procedure. cDNA was synthesized with total RNA and Reverse Transcript PCR kit (Qiagen, Germany), along with the protocol provided. The PCR products were amplified using degenerated primers designed based on the amino acid residues of the N-terminal sequencing and oligo-(dT) 18 V primer (Supplementary Table S1). After a step at 50 °C for 30 min and 95 °C for 15 min, the reaction was performed for 35 cycles (95 °C for 0.5 min, 47 °C for 0.5 min, 72 °C for 1 min), followed by a final step of 7 min at 72 °C.
Crude PCR products (both about 500 bp) were then ligated into a pGEM-T (Promega, USA) vector. After transformation of Escherichia coli XL-1 Blue competent cells with the ligation product, positive colonies were selected by PCR using the plasmid's primers SP6 and T7, then extracted and sequenced. Analysis of five colonies for each sample gave the same sequences. Phylogenetic tree construction. The insect OBP amino acid sequences were analyzed using MUSCLE alignment default settings through MEGA version 6.06 63 (http://www.megasoftware.net/). The phylogenic tree was then generated by the neighbor-joining algorithm since the p-distance was < 0.8 in overall distance 64 . A total of 2,000 bootstrap replications were used to test of phylogeny. Finally, the selected tree was created with the cut-off value of 50%.
Heterologous expression and purification of recombinant CjapOBP proteins. pGEM plasmids containing sequences of the mature CjapOBPs were digested with NdeI and BamHI restriction enzymes. Then the products were ligated into a pET28a vector (Novagen, Germany) linearised with the same enzymes. The recombinant pET-CjapOBP expression plasmid was transformed into E. coli BL21 (DE3) competent cells. Single colonies were grown overnight in 4 ml of Luria-Bertani (LB) broth supplemented with 50 µ g/ml kanamycin at 37 °C with shaking at 200 rpm. The culture was diluted 1:100 with fresh LB broth (supplemented with 50 µ g/ml kanamycin) and grown at 37 °C with shaking at 200 rpm until the OD 600 reached approximately 0.5. Isopropyl β -D-1-thiogalactopyranoside (IPTG) (Merck, Germany) was then added to the culture to a final concentration of 0.4 mM to induce expression of the target products for approximately 3 h at 37 °C. After sonication of the bacterial pellet and centrifugation, all proteins were present in the supernatant, and the recombinant CjapOBP protein were purified using a Ni-NTA His-Bind column following the provided protocol. (Novagen, Germany).

Preparation of antisera.
Polyclonal antisera against CjapOBP1 and CjapOBP2 were obtained by injecting rabbits subcutaneously and intramuscularly with 500 µ g of purified recombinant protein, followed by three additional injections of 300 µ g after 10 days each time. The protein was emulsified with an equal volume of Freund's complete adjuvant (Sigma, St. Louis, MO, USA) for the first injection and incomplete adjuvant (Sigma, St. Louis, MO, USA) for further injections. The blood of rabbits was taken 7 days after the last injection, and centrifuged at 6000 rpm for 30 min. The supernatant of the serum was used without further purification. Rabbits were individually housed in large cages, at constant temperature, and all operations were performed according to ethical guidelines in order to minimize pain and discomfort to animals. The immunizations were performed according to the protocol approved 65 Western blot analysis. Extracts were prepared from antennae, mouth parts, tarsi, sex organs of adults of both sexes, as described in the Section of "Identification of C. japonicus OBPs" in "Materials and Methods". After electrophoretic separation, gels were electroblotted onto nitrocellulose (NC) membrane (Millipore, USA) using a semi-dry protocol 67 . Following overnight treatment with 2% powdered skimmed milk, the membrane was incubated with the crude antiserum at a dilution of 1:500 (2 h) and then with Goat Anti-Rabbit IgG (H+ L), Horseradish Peroxidase Conjugate (Invitrogen, USA) (a dilution of 1:1000 for 1 h). Immunoreacting bands were detected by treatment with 4-chloro-1-naphthol and hydrogen peroxide.
Binding assays. To measure the affinity of the fluorescent ligand N-phenyl-1-naphthylamine (1-NPN), a 2 µ M solution of the protein in 50 mM Tris-HCl (pH 7.4) was titrated with aliquots of 1 mM 1-NPN in methanol to final concentrations of 1-16 µ M. The probe was excited at 337 nm, and emission spectra were recorded between 380 and 450 nm. The affinity of other ligands was measured in competitive binding assays, using the protein and the fluorescent reporter 1-NPN at the concentration of 5 µ M and 2 µ M respectively, while increasing the final concentration of each competitor up to 16 µ M. Dissociation constants for 1-NPN were evaluated from Scatchard plots of the binding data, and other ligands were calculated from the corresponding IC 50