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Molecular Characterization of MbraOR16, a Candidate Sex Pheromone Receptor in Mamestra brassicae (Lepidoptera: Noctuidae)

Abstract Sex pheromone communication in Lepidoptera has long been a valuable model system for studying fundamental aspects of olfaction and its study has led to the establishment of environmental-friendly pest control strategies. The cabbage moth, Mamestra brassicae (Linnaeus) (Lepidoptera: Noctuidae), is a major pest of Cruciferous vegetables in Europe and Asia. Its sex pheromone has been characterized and is currently used as a lure to trap males; however, nothing is known about the molecular mechanisms of sex pheromone reception in male antennae. Using homology cloning and rapid amplification of cDNA ends-PCR strategies, we identified the first candidate pheromone receptor in this species. The transcript was specifically expressed in the antennae with a strong male bias. In situ hybridization experiments within the antennae revealed that the receptor-expressing cells were closely associated with the olfactory structures, especially the long trichoid sensilla known to be pheromone-sensitive. The deduced protein is predicted to adopt a seven-transmembrane structure, a hallmark of insect odorant receptors, and phylogenetically clustered in a clade that grouped a majority of the Lepidoptera pheromone receptors characterized to date. Taken together, our data support identification of a candidate pheromone receptor and provides a basis for better understanding how this species detects a signal critical for reproduction.

Thousands of volatile compounds hover in our environment. For a nocturnal moth, some of them carry crucial information about the host plants or conspecific mates. In moths, as in many insects, mate recognition usually relies on sex pheromone emission and reception by the corresponding partners (Tamaki 1985). Moth sex pheromones are classified into different types, based on the chemical features and biosynthetic pathways of the molecules (Löfstedt et al. 2016). These molecules are present only in picomolar concentrations in the atmospheric mixture, necessitating a highly sensitive and specific detection system (Kaissling 2004, Vogt 2005. This system consists of pheromone-sensitive sensory neurons housed in sensilla located on the antennae (Montagné et al. 2015). Most moth sex pheromone components are hydrophobic and are thought to be transported from the air through the sensillum lymph to the neuron by dedicated proteins, the pheromone-binding proteins (PBPs) (Vogt 2005, Pelosi et al. 2014. Reaching the neuron dendritic membrane, the pheromone components are detected by specific subtypes of odorant receptors (ORs) termed pheromone receptors (PRs). The PRs are delicately tuned to detect the components of the pheromone blend emitted by the conspecific female (Zhang and Löfstedt 2015). As ORs, PRs are also seven transmembrane domain receptors and form heteromeric complexes of unknown stoichiometry with the OR co-receptor (Orco) that is conserved among insects (Larsson et al. 2004, Jones et al. 2005. PRs tuned to type I pheromone components all belong to the same OR subfamily, but some PRs tuned to type 0 and type II pheromone components have been recently described in other lepidopteran OR subfamilies (Li et al. 2017, Yuvaraj et al. 2017. Pheromones have been used for decades for environmental-friendly pest management and control (Witzgall et al. 2010); thus, a better understanding of the molecular process of pheromone detection would help improve these strategies. Especially, identification of sex PRs in crop pests opens the way to act as early as the reception step for signal disruption, via the search of receptor activators and/or inhibitors that would interfere with the receptor Journal of Insect Science, (2018) 18(5): 5; 1-8 doi: 10.1093/jisesa/iey090 Short Communication response. More avenues of effective mating disruption could become available with the identification, via OR functional screening, of behavior modifying chemicals such as host plant volatiles for mating sites (push-pull strategies) (Cook et al. 2007) or intervening in host seeking strategies for feeding and/or egg laying (Conchou et al. 2017 (Hill 1987). Its sex pheromone has been characterized (Attygalle et al. 1987) and is currently used as a lure to trap males, but very little is known about the molecular mechanisms of sex pheromone reception in male antennae. Although these mechanisms are well studied in many moths as reviewed (Zhang and Löfstedt 2015), it is surprising that no PR has yet been characterized in M. brassicae. So far, only Orco (Malpel et al. 2008) and two PBPs have been cloned in this species (Maïbèche-Coisné et al. 1998). To extend the sex pheromone reception cascade in M. brassicae, here we present multiple lines of evidence to suggest that MbraOR16 is a PR expressed in the olfactory sensilla of male M. brassicae antennae.

Insect Rearing and cDNA Synthesis
Insects were reared on a semiartificial diet (Poitout and Buès 1974) at 20°C, 60-70% relative humidity and under a 16:8 light:dark cycle. For cDNA synthesis, various tissues (male and female antennae, proboscis, brain-suboesophageal ganglion complex, thorax, abdomens, legs, and wings) were dissected from 3-d-old adults. For in situ hybridization, male antennae were cut into pieces and fixed overnight in 4% paraformaldehyde (PFA) at 4°C, then dehydrated in methanol, and stored at −20°C until use. Total RNAs were extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). Single-stranded cDNAs were synthesized from 1-μg total RNA for each sample after DNaseI treatment (Promega, Madison WI) with M-MLV reverse transcriptase and oligo(dT) primer using protocol supplied in the Advantage RT-for-PCR kit (Clontech, Mountain View, CA) and were used as template for PCR reactions. For 5′ and 3′ rapid amplification of cDNA ends (RACE), 3′-and 5′-cDNAs were synthesized from 1-μg total RNA from male antennae by using the SMART RACE cDNA Amplification kit (Clontech) according to the manufacturer's instructions.

Phylogeny
The MbraOR16 sequence was aligned with 66 PR sequences from 16 Lepidoptera species (details in Fig. 3) using MAFFT v.7 (Katoh and Standley 2013). The 66 sequences included in the dataset belong to the so-called 'pheromone receptor clade', which includes most of the PRs identified to date that are tuned to type I pheromone components. Phylogenetic reconstruction was performed with PhyML 3.0 (Guindon et al. 2010) using the maximum likelihood method with the JTT + I + G + F substitution model (Jones et al. 1992) and both SPR (Subtree Pruning and Regrafting) and NNI (Nearest Neighbor Interchange) methods for topology improvement. Rate heterogeneity was set at four categories, and values calculated by ProtTest were used for the gamma distribution parameter and the proportion of invariable sites. Node support was estimated using a hierarchical likelihood-ratio test (Anisimova and Gascuel 2006).

Molecular Cloning of MbraOR16 Full-Length cDNA
With primers designed to conserved regions of noctuid PRs, we were able to amplify a fragment of a M. brassicae OR (Fig. 1). Subsequent RACE PCRs facilitated identification of a full-length cDNA encoding a 430-amino acid protein with high homology (72% identity and 84% similarity on average) to previously identified ORs annotated as PRs in various moth species (Fig. 2). The cDNA sequence has been deposited in GenBank (accession number: MF431269). As expected for insect ORs, the sequence was predicted to have seven transmembrane domains (Fig. 1). The C-terminal region known to be conserved in moth PRs was present as were the three highly conserved PR motifs (Fig. 2) (Zhang and Löfstedt 2015).

Phylogenetic Analysis
We constructed a maximum-likelihood phylogeny of the OR subfamily members that have been reported to be tuned to type I pheromones. In this tree, sequences grouped within five distinct clades (named A to E in Fig. 3) that were highly supported by the likelihood-ratio test. PRs from Noctuidae clustered only in clades D and E. MbraOR16 belonged to a particular lineage within clade E that included PR sequences (OR6 and OR16) from H. armigera, Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae), Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), and Agrotis segetum (Schiff.) (Lepidoptera: Noctuidae) that are tuned to type I pheromones of different natures. Due to moderate support at several nodes within this lineage, the precise phylogenetic position of MbraOR16 could not be determined.
in expression across the various tissues investigated (Fig. 4, lower part).

Antennal Expression Pattern via In Situ Hybridization
M. brassicae antennae are filiform and segmented, and their dorsal part is covered with scales, whereas the ventral part has numerous trichoid sensilla (Jacquin-Joly et al. 2000) devoted to olfaction. Although no signal was generated with the sense probe (Fig. 5A), antisense probe labeling was restricted to the ventral side (the olfactory side) of the male antennae (Fig. 5B). More precisely, MbraOR16 transcripts were localized to olfactory sensilla bases in the male antenna, which likewise house olfactory neuron cell bodies. Expression in particular could be seen at the bases of the long trichoid sensilla, which are known to be involved in pheromone reception and are easily recognizable in the transversal sections of the antennal segments (Fig. 5C).

Discussion
Our results suggest that we cloned a full-length mRNA encoding MbraOR16, which represents the first candidate PR of the crop pest moth M. brassicae. The encoded protein possessed the hallmarks of insect ORs, such as seven transmembrane domains and specific expression in the antennae that in situ hybridization showed was restricted to olfactory sensilla (Fig. 5). Additional findings support classification of this receptor as a PR: 1) the transcript was male enriched, as usually observed for moth PRs Löfstedt 2015, Zhang et al. 2015); 2) the deduced protein possessed the conserved C-terminal signature motifs typical of moth PRs (Zhang and Löfstedt 2015); and 3) MbraOR16 clustered in a lineage containing noctuid PRs tuned to type I pheromone components. Taken together, our findings suggest that MbraOR16 is also a type I PR, whereas M. brassicae pheromone blend consists of type I components. However, the precise position of MbraOR16 within the clade could not be determined. Furthermore, because PR response spectra evolved rapidly, it is difficult to infer a putative ligand for MbraOR16 based on the phylogeny. Nevertheless, several lines of evidence let us propose that we have identified the receptor for M. brassicae behavioral antagonists Z11-16:OH and/or Z9-14:Ac (Descoins et al. 1978), compounds found in the sex blends of heterospecific females that prevent unspecific attraction between species with similar sex pheromone blends. The in situ hybridization performed on male antennae indicates that MbraOR16 transcripts were localized in long trichoid sensilla, that are known to house two neurons, one tuned to the main pheromone component Z11-16:Ac, and the other one tuned to the behavioral antagonists Z11-16:OH and Z9-14:Ac (Renou 1991, Renou andLucas 1994). In the phylogeny, MbraOR16 did not cluster with moth PRs tuned to Z11-16:Ac, such as H. virescens OR14 (Wang et al. 2011) and Mythimna separata (Walker) (Lepidoptera: Noctuidae) OR1 (Mitsuno et al. 2008), but rather clustered with receptors tuned to Z11-16:OH, such as H. armigera and H. virescens OR16 (Wang et al. 2011, Liu et al. 2013b, and to Z9-14:Ac, such as A. segetum OR10 (Zhang and Löfstedt, 2013). However, unequivocal demonstration of ligand tuning will require further functional studies for a better understanding of M. brassicae sex pheromone reception.   4. RT-PCR expression study of MbraOR16 in samples derived from different tissues. PCR products are visualized by ethidium bromide after electrophoresis on a 1.5% agarose gel. Tissues examined included male antennae, female antennae, proboscis, Br-SOG (brain-subesophageal ganglion complex), thorax, abdomens, legs, and wings. MbraOR16 amplification led to a 765 bp product. A 508-bp fragment of ribosomal protein 8 (rpl8) was amplified in each sample and used as a cDNA integrity control. Ladder: 1kb DNA ladder (Invitrogen). Only the 500-bp band is visible.