Identification of candidate chemosensory genes by transcriptome analysis in Loxostege sticticalis Linnaeus

Abstract

Loxostege sticticalis Linnaeus is an economically important agricultural pest, and the larvae cause great damage to crops, especially in Northern China. However, effective and environmentally friendly chemical methods for controlling this pest have not been discovered to date. In the present study, we performed HiSeq2500 sequencing of transcriptomes of the male and female adult antennae, adult legs and third instar larvae, and we identified 54 candidate odorant receptors (ORs), including 1 odorant receptor coreceptor (Orco) and 5 pheromone receptors (PRs), 18 ionotropic receptors (IRs), 13 gustatory receptors (GRs), 34 odorant binding proteins (OBPs), including 1 general odorant binding protein (GOBP1) and 3 pheromone binding proteins (PBPs), 10 chemosensory proteins (CSPs) and 2 sensory neuron membrane proteins (SNMPs). The results of RNA-Seq and RT-qPCR analyses showed the expression levels of most genes in the antennae were higher than that in the legs and larvae. Furthermore, PR4, OR1-4, 7-11, 13-15, 23, 29-32, 34, 41, 43, 47/IR7d.2/GR5b, 45, 7/PBP2-3, GOBP1, OBP3, 8 showed female antennae-biased expression, while PR1/OBP2, 7/IR75d/CSP2 showed male antennae-biased expression. However, IR1, 7d.3, 68a/OBP11, 20-22, 28/CSP9 had larvae enriched expression, and OBP15, 17, 25, 29/CSP5 were mainly expressed in the legs. The results shown above indicated that these genes might play a key role in foraging, seeking mates and host recognition in the L. sticticalis. Our findings will provide the basic knowledge for further studies on the molecular mechanisms of the olfactory system of L. sticticalis and potential novel targets for pest control strategies.

Fig 1. Gene ontology classified annotation of the L. sticticalis unigenes.

Fig 2. Phylogenetic tree of candidate LstiORs with known lepidopteran ORs.

Csup: C. suppressalis, Bmor: B. mori, Harm: H. armigera, Hass: H. assulta. The clade in blue indicates the PR gene clade; the clade in pink indicates the Orco clade.

Fig 3. Expression pattern of L. sticticalis ORs by RT-qPCR.

Legs (male: female = 1:1). β-actin was used as an internal reference gene to test the integrity of each cDNA template. The standard error is represented by the error bar, and the different letters (a, b, c) above each bar represent significant differences (p < 0.05).

Fig 4. Phylogenetic tree of candidate LstiIRs with known lepidopteran IRs and iGluRs.

Dmel: D. melanogaster, Bmor: B. mori, Slit: S. littoralis. The clade in blue indicates the iGluR gene clade; the clade in pink indicates the IR8a and IR25a clade.

Fig 5. Expression pattern of L. sticticalis IRs by RT-qPCR.

The details were same as mentioned in Fig 3.

Fig 6. Phylogenetic tree of candidate LstiGRs with known lepidopteran GRs.

Dmel: D. melanogaster, Bmor: B. mori, Harm: H. armigera and Hass: H. assulta.

Fig 7. Expression pattern of L. sticticalis GRs by RT-qPCR.

The details were same as mentioned in Fig 3.

Fig 8. Phylogenetic tree of candidate LstiOBPs with known lepidopteran OBPs.

Csup: C. suppressalis, Bmor: B. mori, Harm: H. armigera, Hass: H. assulta, Cpun: C. punctiferalis. The clade in blue indicates the GOBP gene clade; the clade in pink indicates the PBP clade.

Fig 9. Expression pattern of L. sticticalis OBPs by RT-qPCR.

The details were same as mentioned in Fig 3.

Fig 10. Phylogenetic tree of candidate LstiCSPs with known lepidopteran CSPs.

Csup: C. suppressalis, Cpun: C. punctiferalis, Bmor: B. mori, Harm: H. armigera.

Fig 11. Expression pattern of L. sticticalis CSPs by RT-qPCR.

The details were same as mentioned in Fig 3.

Fig 12. Phylogenetic tree of candidate LstiSNMPs with known lepidopteran SNMPs.

Onub: O. nubilalis, Csup: C. suppressalis, Cmed: C. medinalis, Hvir: Heliothis viresscens, Sexi: S. exigua, Slit: S. litura, Msex: Manduca sexta.

Fig 13. Comparative results of olfactory genes FPKM in the male antennae, female antennae, legs and third instar larvae of L. sticticalis (Venn diagram).

A. comparison among the antennae, legs and larvae. B. comparison between the male and female antennae. Genes in the overlapping intersect show no significant difference among different tissues. Genes outside the intersect show significant difference. Those in the dash-outlined area show specific expression in the tissues.