Identification and functional analysis of olfactory receptor family reveal unusual characteristics of the olfactory system in the migratory locust

Abstract

Locusts represent the excellent model of insect olfaction because the animals are equipped with an unusual olfactory system and display remarkable density-dependent olfactory plasticity. However, information regarding receptor molecules involved in the olfactory perception of locusts is very limited. On the basis of genome sequence and antennal transcriptome of the migratory locust, we conduct the identification and functional analysis of two olfactory receptor families: odorant receptors (ORs) and ionotropic receptors (IRs). In the migratory locust, there is an expansion of OR family (142 ORs) while distinctly lower number of IR genes (32 IRs) compared to the repertoires of other insects. The number of the locust OR genes is much less than that of glomeruli in antennal lobe, challenging the general principle of the "one glomerulus-one receptor" observed in other insects. Most OR genes are found in tandem arrays, forming two large lineage-specific subfamilies in the phylogenetic tree. The "divergent IR" subfamily displays a significant contraction, and most of the IRs belong to the "antennal IR" subfamily in the locust. Most ORs/IRs have olfactory-specific expression while some broadly- or internal-expressed members are also found. Differing from holometabolous insects, the migratory locust contains very similar expression profiles of ORs/IRs between nymph and adult stages. RNA interference and behavioral assays indicate that an OR-based signaling pathway, not IR-based, mediates the attraction of locusts to aggregation pheromones. These discoveries provide insights into the unusual olfactory system of locusts and enhance our understanding of the evolution of insect olfaction.

Keywords: Aggregative behavior; Chemoreceptor; Hemimetabolous; Orthoptera; Peripheral olfactory system; Phase polyphenism.

Fig. 1

Genomic locations of partial LmigOR genes. The central lines represent two of scaffolds assembled in the locust genome. The orientation of gene transcription is shown with an arrow. The scaffold length (kb), gene locations and orientations are based on data from Release 2.4 of locust genome. AA amino acid

Fig. 2

Phylogenetic analysis of LmigORs. 126 LmigORs (AA length > 250) were selected to build the tree. The dendrogram was generated by Bayesian analysis (WAG substitution model) and RAxML (JTT substitution model). Only support values for major branches and above 50 % are shown. The value before the solidus is given by Bayesian analysis whereas that after the solidus is given by RAxML method. Suffixes after OR names: P pseudogene, N N-terminal missing, C C-terminal missing, I internal region missing. Species abbreviations: Amel, Apis mellifera; Apis, Acyrthosiphon pisum; Bmor, Bombyx mori; Dmel, Drosophila melanogaster; Agam, Anopheles gambiae; Lmig, Locusta migratoria. The scale bar represents the expected changes per site

Fig. 3

iGluRs/IRs identified in the locust. a Phylogenetic analysis of iGluRs/IRs. The dendrogram was generated using Bayesian analysis (WAG substitution model). Only support values for major branches are shown. Sequences of Daphnia pulex, A. pisum, and D. melanogaster are taken from reference 16; Z. nevadensis sequences are taken from reference 39. The scale bar represents the expected changes per site. b Histogram of the number of iGluR, antennal IR, and divergent IR genes identified in different species. The gene number of the different sub-families is counted according to reference 16 except for P. siccifolium, which is counted according to reference 5. The gene numbers in Z. nevadensis and L. migratoria are counted according to our in-house phylogenetic analysis. The species names involved in phylogenetic tree building are colored, and the color pattern is consistent with that in the tree. The organisms are sorted according to the evolutionary status. Filled triangle, genome is not sequenced

Fig. 4

Expression of LmigOR genes in different adult tissues. a Left Heat map of LmigOR transcript abundances expressed in different adult tissues. Right Expanded view of the ORs expressed predominantly in internal tissues. b Expression of OR transcripts was confirmed by RT-PCR. The tissue samples were dissected from gregarious adults. c Comprehensive list of LmigORs expressed in adult and nymphal antennae RNAseq. 133, the number of ORs detected in both stages. d RT-PCR analysis of development-specific LmigORs in the adult and nymph antennae

Fig. 5

Behavioral changes of gregarious nymphs after RNAi. a–c Effects of RNAi of LmigOrco, LmigIR8a, and LmigIR25a genes (n = 4 or 6). Data conformed to a normal distribution as checked by the Shapiro–Wilk test, and statistical difference was evaluated by two-tailed Student’s t-test assuming unequal variance. d Dual-choice of gregarious nymphs in Y-tube olfactometer after injection of dsGFP, dsLmigOrco, or dsLmigIR8a/LmigIR25a (n = 57, 65, 56, respectively). Attraction index = (N _v − N _a)/N _v + N _a + N _nc; N _v, the number of “choose volatiles”; N _a, the number of “choose air”; N _nc, the number of “no choice”. Statistical difference was evaluated by Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. Data are mean ± SEM