Identification of candidate chemosensory genes in the antennal transcriptome of Monolepta signata

Abstract

In the polyphagous insect Monolepta signata (M. signata) (Coleoptera: Chrysomelidae), antennae are important for olfactory reception used during feeding, mating, and finding a suitable oviposition site. Based on NextSeq 6000 Illumina sequencing, we assembled the antennal transcriptome of mated M. signata and described the first chemosensory gene repertoire expressed in this species. The relative expression levels of some significant chemosensory genes were conducted by quantitative real-time PCR. We identified 114 olfactory-related genes based on the antennal transcriptome database of M. signata, including 21 odorant binding proteins (OBPs), six chemosensory proteins (CSPs), 46 odorant receptors (ORs), 15 ionotropic receptors (IRs), 23 gustatory receptors (GRs) and three sensory neuron membrane proteins (SNMPs). Blastp best hit and phylogenetic analyses showed that most of the chemosensory genes had a close relationship with orthologs from other Coleoptera species. Overall, this study provides a foundation for elucidating the molecular mechanism of olfactory recognition in M. signata as well as a reference for the study of chemosensory genes in other species of Coleoptera.

Fig 1. Multiple amino acid sequence alignment of Classic OBPs in M. signata.

The six conserved cysteine residues are highlighted in black ground and red border. Amino acids that are more than 50% identical in all sequences are marked with cyan color highlights, and more than 75% identical are marked with pink highlights.

Fig 2. Multiple amino acid sequence alignment of Minus-C OBPs in M. signata.

The four conserved cysteine residues are highlighted in black ground and red border. Amino acids that are more than 50% identical in all sequences are marked with cyan color highlights, and more than 75% identical are marked with pink highlights.

Fig 3. Phylogenetic tree of candidate MsigOBPs with known Coleopteran OBP sequences.

Amal, Agrilus mali (N = 2); Cbow, Colaphellus bowringi (N = 25); Hobl, Holotrichia oblita (N = 9); Hpar, Holotrichia parallela (N = 3); Malt, Monochamus alternatus (N = 7); Pyas, Pachyrhinus yasumatsui (N = 2); Szea, Sitophilus zeamais (N = 2); Tcas, Tribolium castaneum (N = 48). Bootstrap values >50 are shown.

Fig 4. Multiple amino acid sequence alignment of CSPs in M. signata.

The four conserved cysteine residues are highlighted in black ground and red border. Amino acids that are more than 50% identical in all sequences are marked with cyan color highlights, and more than 75% identical are marked with pink highlights.

Fig 5. Phylogenetic tree of candidate MsigCSPs with known Coleopteran CSP sequences.

Amal, Agrilus mali (N = 4); Bhor, Batocera horsfieldi (N = 1); Cfor, Cylas formicarius (N = 3); Darm, Dendroctonus armandi (N = 9); Hpar, Holotrichia parallela (N = 1); Lory, Lissorhoptrus oryzophilus (N = 5); Malt, Monochamus alternatus (N = 12); Ocom, Ophraella communa (N = 12); Pyas, Pachyrhinus yasumatsui (N = 1); Tcas, Tribolium castaneum (N = 18). Bootstrap values >50 are shown.

Fig 6. Phylogenetic tree of candidate MsigORs with known Coleopteran OR sequences.

Achi, Anoplophora chinensis (N = 1); Agla, Anoplophora glabripennis (N = 1); Aqua, Ambrostoma quadriimpressum (N = 1); Cbow, Colaphellus bowringi (N = 31); Cchi, Callosobruchus chinensis (N = 4); Dvir, Diabrotica virgifera virgifera (N = 1); Gdau, Galeruca daurica (N = 1); Habi, Hylobius abietis (N = 2); Hobl, Holotrichia oblita (N = 4); Ityp, Ips typographus (N = 5); Malt, Monochamus alternatus (N = 1); Ocom, Ophraella communa (N = 1); Otau, Onthophagus taurus (N = 1);Pbre, Protaetia brevitarsis (N = 1); Pver, Plagiodera versicolora (N = 1); Rfer, Rhynchophorus ferrugineus (N = 1); Rvul, Rhynchophorus vulneratus (N = 1); Svel, Sympiezomias velatus (N = 1); Tcas, Tribolium castaneum (N = 91); Tmol, Tenebrio molitor (N = 1). Bootstrap values >50 are shown.

Fig 7. Phylogenetic tree of candidate MsigIRs with known Coleopteran IR sequences.

Blon, Brontispa longissima (N = 13); Bmel, Basilepta melanopus (N = 15); Cbow, Colaphellus bowringi (N = 7); Cfor, Cylas formicarius (N = 13); Dpon, Dendroctonus ponderosae (N = 5); Otau, Onthophagus taurus (N = 3); Pver, Plagiodera versicolora (N = 4); Tcas, Tribolium castaneum (N = 33); Tmol, Tenebrio molitor (N = 7). Bootstrap values >50 are shown.

Fig 8. Phylogenetic tree of candidate MsigGRs with known Coleopteran GR sequences.

Dmel, Drosophila melanogaster(N = 67); Pstr, Phyllotreta striolata (N = 12); Tcas, Tribolium castaneum (N = 11). Bootstrap values >50 are shown.

Fig 9. Phylogenetic tree of candidate MsigSNMPs with known Coleopteran SNMP sequences.

Achi, Anoplophora chinensis (N = 3); Bmel, Basilepta melanopus (N = 4); Cbow, Colaphellus bowringi (N = 4); Cfor, Cylas formicarius (N = 3); Dpon, Dendroctonus ponderosae (N = 3); Dvir, Diabrotica virgifera virgifera (N = 1); Gdau, Galeruca daurica (N = 2); Ityp, Ips typographus (N = 2); Ocom, Ophraella communa (N = 4); Paen, Pyrrhalta aenescens (N = 2); Pmac, Pyrrhalta maculicollis (N = 2); Pstr, Phyllotreta striolata (N = 2); Pver, Plagiodera versicolora (N = 4); Tcas, Tribolium castaneum (N = 2).Bootstrap values > 50 are shown.

Fig 10. The relative expression levels of OBPs in male and female antennae of M. signata by RT-qPCR.

FA: female antennae; MA: male antennae. The GAPDH gene was used to normalize expression levels in each sample, and the female antennae were selected as the calibrator to normalize the gene expression levels in various tissues. ** Indicates that the difference is extremely significant (P<0.01), * indicates that the difference is significant (P<0.05), ns means no significant difference (P>0.05). Standard errors are represented by the error bars.

Fig 11. The relative expression levels of CSPs in male and female antennae of M. signata by RT-qPCR.

FA: female antennae; MA: male antennae. The GAPDH gene was used to normalize expression levels in each sample, and the female antennae were selected as the calibrator to normalize the gene expression levels in various tissues. ** Indicates that the difference is extremely significant (P<0.01), * indicates that the difference is significant (P<0.05), ns means no significant difference (P>0.05). Standard errors are represented by the error bars.

Fig 12. The relative expression levels of ORs in male and female antennae of M. signata by RT-qPCR.

FA: female antennae; MA: male antennae. The GAPDH gene was used to normalize expression levels in each sample, and the female antennae were selected as the calibrator to normalize the gene expression levels in various tissues. ** Indicates that the difference is extremely significant (P<0.01), * indicates that the difference is significant (P<0.05), ns means no significant difference (P>0.05). Standard errors are represented by the error bars.