Molecular characterization and expression of sensory neuron membrane proteins in the parasitoid Microplitis mediator (Hymenoptera: Braconidae)

Abstract

Sensory neuron membrane proteins (SNMPs), homologs of the human fatty acid transport protein CD36 family, are observed to play a significant role in chemoreception, especially in detecting sex pheromone in Drosophila and some lepidopteran species. In the current study, two full-length SNMP transcripts, MmedSNMP1 and MmedSNMP2, were identified in the parasitoid Microplitis mediator (Hymenoptera: Braconidae). Quantitative real-time polymerase chain reaction analysis showed that the expression of MmedSNMP1 was significantly higher in antennae than in other tissues of both sexes. In addition, the MmedSNMP1 transcript was increased dramatically in newly emerged adults and there were no significant differences between adults with or without mating and parasitic experiences. However, compared with MmedSNMP1, the expression of MmedSNMP2 was widely found in various tissues, significantly increased at half-pigmented pupae stage and remained at a relatively constant level during the following developmental stages. It was found that MmedSNMP1 contained eight exons and seven introns, which was highly conserved compared with other insect species. In situ hybridization assay demonstrated that MmedSNMP1 transcript was distributed widely in antennal flagella. Among selected chemosensory genes (odorant binding protein, odorant receptor, and ionotropic receptor genes), MmedSNMP1 only partially overlapped with MmedORco in olfactory sensory neurons of antennae. Subsequent immunolocalization results further indicated that MmedSNMP1 was mainly expressed in sensilla placodea of antennae and possibly involved in perceiving plant volatiles and sex pheromones. These findings lay a foundation for further investigating the roles of SNMPs in the chemosensation of parasitoids.

Keywords: Microplitis mediator; antennae; chemosensation; expression profile; in situ hybridization; sensory neuron membrane proteins.

Figure 1

Phylogenetic analysis of sensory neuron membrane proteins (SNMPs) from different insect orders. This neighbor‐joining tree is constructed using MEGA 5 with a p‐distance model and pairwise by resampling amino acid position 1000 times. The SNMP sequences used in this analysis are listed in Supplemental material 1.

Figure 2

Expression profiles of MmedSNMP1 (A) and MmedSNMP2 (B) in different tissues. An: antennae, He: heads, Th: thoraxes, Ab: abdomens, Le: legs, Wi: wings. The fold changes are relative to the transcript levels in the abdomens of both sexes. The different small letters above each bar indicate significant differences in SNMP transcript levels of different tissues (P < 0.05). The asterisks represent significant differences in expression levels of SNMPs between males and females. (*, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, 0.0001 < P < 0.001, ns, no significant difference).

Figure 3

Expressions of MmedSNMP1 and MmedSNMP2 at different developmental stages and physiological states. (A–D) The fold changes are relative to the sensory neuron membrane protein (SNMP) transcript levels in antennae of Microplitis mediator at red‐eyed stage. The different small letters above each bar indicate significant differences in transcript abundances using Duncan's multiple range test (P < 0.05). (E) Schematic photos of M. mediator at different developmental stages and experience states. (a) Red‐eyed stage, (b) half‐pigmented stage, (c) fully pigmented stage, (d) newly emerged adult, (e) unmated adult, (f) mated adult without parasitic experience, (g) mated adult with parasitic experience.

Figure 4

Gene structure and intron insertion sites analysis of sensory neuron membrane protein 1 (SNMP1). (A) Gene structure of MmedSNMP1. Exons are showed by black round‐corner rectangle and introns are showed by black line. (B) Comparison of intron insertion sites of SNMP1s among selected insect species. Long vertical bars show the introns insert position, short vertical bars show the initial and final amino acid positions. Numbers above indicate the insertion site character positions, while numbers in parentheses indicate the phase of each intron insertion site. Mmed: Microplitis mediator; Mdem: M. demolitor; Nvit: Nasonia vitripennis; Amel: Apis mellifera; Dmel: Drosophila melanogaster; Dpse: D. pseudoobscura; Aaeg: Aedes aegypti; Agam: Anopheles gambiae; Bmor: Bombyx mori; Tcas: Tribolium castaneum.

Figure 5

In situ hybridization assay of SNMP1 in antennae of Microplitis mediator. Digoxigenin‐labeled antisense RNA probes for SNMP1 are hybridized to cell clusters in longitudinal sections of antennal flagella segments of female (A, B) and male (C, D). Signals are visualized by red fluorescence.

Figure 6

Localization of SNMP1 in antennae of male Microplitis mediator. MmedSNMP1 (green) is expressed in long axis (A–C), central dendritic bundle channel (D–F) and double ridges (G, H) of sensilla placodea. (I) Morphology of s. placodea (Wang et al., 2018). White arrows (H, I) indicate the morphology of s. placodea. Black arrow (I) indicates the transverse section of s. placodea where there are multiple pores on the wall. (J) Western blot analysis of the MmedSNMP1.

Figure 7

Co‐localization of MmedSNMP1 and MmedOrco in antennae of Microplitis mediator. Combined DIG‐labeled and biotin‐labeled probes of MmedSNMP1 and MmedOrco are hybridized to vertical and horizontal sections of antennae from both sexes. Left panel shows the MmedSNMP1, middle panel shows the MmedOrco, and right panel shows merged pictures.

Figure 8

Double fluorescence in situ hybridization assays of MmedSNMP1 with chemosensory genes in antennae of Microplitis mediator. DIG‐labeled and biotin‐labeled probes of MmedSNMP1 and MmedIR8a/IR25a1/OBP3/OBP2 are hybridized to vertical and horizontal sections of antennae. White dashed frame areas are illustrated at a higher magnification on the right.