Christine Merlin

The Monarch Butterfly Genome Yields Insights into Long-Distance Migration

We present the draft 273 Mb genome of the migratory monarch butterfly (Danaus plexippus) and a set of 16,866 protein-coding genes. Orthology properties suggest that the Lepidoptera are the fastest evolving insect order yet examined. Compared to the silkmoth Bombyx mori, the monarch genome shares prominent similarity in orthology content, microsynteny, and protein family sizes. The monarch genome reveals a vertebrate-like opsin whose existence in insects is widespread; a full repertoire of molecular components for the monarch circadian clockwork; all members of the juvenile hormone biosynthetic pathway whose regulation shows unexpected sexual dimorphism; additional molecular signatures of oriented flight behavior; microRNAs that are differentially expressed between summer and migratory butterflies; monarch-specific expansions of chemoreceptors potentially important for long-distance migration; and a variant of the sodium/potassium pump that underlies a valuable chemical defense mechanism. The monarch genome enhances our ability to better understand the genetic and molecular basis of long-distance migration.

An Expressed Sequence Tag collection from the male antennae of the Noctuid moth Spodoptera littoralis : a resource for olfactory and pheromone detection research

BackgroundNocturnal insects such as moths are ideal models to study the molecular bases of olfaction that they use, among examples, for the detection of mating partners and host plants. Knowing how an odour generates a neuronal signal in insect antennae is crucial for understanding the physiological bases of olfaction, and also could lead to the identification of original targets for the development of olfactory-based control strategies against herbivorous moth pests. Here, we describe an Expressed Sequence Tag (EST) project to characterize the antennal transcriptome of the noctuid pest model, Spodoptera littoralis, and to identify candidate genes involved in odour/pheromone detection.ResultsBy targeting cDNAs from male antennae, we biased gene discovery towards genes potentially involved in male olfaction, including pheromone reception. A total of 20760 ESTs were obtained from a normalized library and were assembled in 9033 unigenes. 6530 were annotated based on BLAST analyses and gene prediction software identified 6738 ORFs. The unigenes were compared to the Bombyx mori proteome and to ESTs derived from Lepidoptera transcriptome projects. We identified a large number of candidate genes involved in odour and pheromone detection and turnover, including 31 candidate chemosensory receptor genes, but also genes potentially involved in olfactory modulation.ConclusionsOur project has generated a large collection of antennal transcripts from a Lepidoptera. The normalization process, allowing enrichment in low abundant genes, proved to be particularly relevant to identify chemosensory receptors in a species for which no genomic data are available. Our results also suggest that olfactory modulation can take place at the level of the antennae itself. These EST resources will be invaluable for exploring the mechanisms of olfaction and pheromone detection in S. littoralis, and for ultimately identifying original targets to fight against moth herbivorous pests.

Antennal Circadian Clocks Coordinate Sun Compass Orientation in Migratory Monarch Butterflies

Butterfly Navigation Monarch butterflies migrate to Mexico from various parts of North America in the fall and navigate with the aid of Sun compass. This navigational mechanism, also employed by migratory birds, uses the circadian clock to compensate for the positional change of the Sun in the sky throughout the day. The mechanism behind time-compensated Sun compass orientation has remained obscure. Merlin et al. (p. 1700; see the Perspective by Kyriacou) now provide comprehensive data showing that the mechanism resides in the antennae of the butterflies, rather than the brain, as previously thought. The “antennal clocks” found in the monarchs probably provide the primary timing mechanism for Sun compass orientation. These findings reveal a further function for the antennae—a function that may extend widely to other insects that use this orientation mechanism. Monarch butterfly antennae contain the timing mechanism for time-compensated Sun compass orientation. During their fall migration, Eastern North American monarch butterflies (Danaus plexippus) use a time-compensated Sun compass to aid navigation to their overwintering grounds in central Mexico. It has been assumed that the circadian clock that provides time compensation resides in the brain, although this assumption has never been examined directly. Here, we show that the antennae are necessary for proper time-compensated Sun compass orientation in migratory monarch butterflies, that antennal clocks exist in monarchs, and that they likely provide the primary timing mechanism for Sun compass orientation. These unexpected findings pose a novel function for the antennae and open a new line of investigation into clock-compass connections that may extend widely to other insects that use this orientation mechanism.

INSECT OLFACTORY RECEPTORS: CONTRIBUTIONS OF MOLECULAR BIOLOGY TO CHEMICAL ECOLOGY

Our understanding of the molecular basis of chemical signal recognition in insects has been greatly expanded by the recent discovery of olfactory receptors (Ors). Since the discovery of the complete repertoire of Drosophilamelanogaster Ors, candidate Ors have been identified from at least 12 insect species from four orders (Coleoptera, Lepidoptera, Diptera, and Hymenoptera), including species of economic or medical importance. Although all Ors share the same G-protein coupled receptor structure with seven transmembrane domains, they present poor sequence homologies within and between species, and have been identified mainly through genomic data analyses. To date, D. melanogaster remains the only insect species where Ors have been extensively studied, from expression pattern establishment to functional investigations. These studies have confirmed several observations made in vertebrates: one Or type is selectively expressed in a subtype of olfactory receptor neurons, and one olfactory neuron expresses only one type of Or. In addition, all olfactory neurons expressing one Or type converge to the same glomerulus in the antennal lobe. The olfactory mechanism, thus, appears to be conserved between insects and vertebrates. Although Or functional studies are in their initial stages in insects (mainly Drosophila), insects appear to be good models to establish fundamental concepts of olfaction with the development of powerful genetic, imaging, and behavioral tools. This new field of study will greatly contribute to the understanding of insect chemical communication mechanisms, particularly with agricultural pests and disease vectors, and could result in future strategies to reduce their negative effects.

Navigational mechanisms of migrating monarch butterflies

Recent studies of the iconic fall migration of monarch butterflies have illuminated the mechanisms behind their southward navigation while using a time-compensated sun compass. Skylight cues, such as the sun itself and polarized light, are processed through both eyes and are probably integrated in the brains central complex, the presumed site of the sun compass. Time compensation is provided by circadian clocks that have a distinctive molecular mechanism and that reside in the antennae. Monarchs might also use a magnetic compass because they possess two cryptochromes that have the molecular capability for light-dependent magnetoreception. Multiple genomic approaches are now being used with the aim of identifying navigation genes. Monarch butterflies are thus emerging as an excellent model organism in which to study the molecular and neural basis of long-distance migration.

An antennal circadian clock and circadian rhythms in peripheral pheromone reception in the moth Spodoptera littoralis.

Circadian rhythms are observed in mating behaviors in moths: females emit sex pheromones and males are attracted by these pheromones in rhythmic fashions. In the moth Spodoptera littoralis, we demonstrated the occurrence of a circadian oscillator in the antenna, the peripheral olfactory organ. We identified different clock genes, period (per), cryptochrome1 (cry1) and cryptochrome2 (cry2), in this organ. Using quantitative real-time PCR (qPCR), we found that their corresponding transcripts cycled circadianly in the antenna as well as in the brain. Electroantennogram (EAG) recordings over 24 h demonstrated for the first time a circadian rhythm in antennal responses of a moth to sex pheromone. qPCR showed that out of one pheromone-binding protein (PBP), one olfactory receptor (OR), and one odorant-degrading enzyme (ODE), all putatively involved in the pheromone reception, only the ODE transcript presented a circadian rhythm that may be related to rhythms in olfactory signal resolution. Peripheral or central circadian clock control of olfaction is then discussed in light of recent data.

Genomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis

The eastern North American monarch butterfly, Danaus plexippus, is an emerging model system to study the neural, molecular, and genetic basis of animal long-distance migration and animal clockwork mechanisms. While genomic studies have provided new insight into migration-associated and circadian clock genes, the general lack of simple and versatile reverse-genetic methods has limited in vivo functional analysis of candidate genes in this species. Here, we report the establishment of highly efficient and heritable gene mutagenesis methods in the monarch butterfly using transcriptional activator-like effector nucleases (TALENs) and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9). Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates, we show that both TALENs and CRISPR/Cas9 generate high-frequency nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites (up to 100%), and that injecting fewer than 100 eggs is sufficient to recover mutant progeny and generate monarch knockout lines in about 3 months. Our study also genetically defines monarch CLK as an essential component of the transcriptional activation complex of the circadian clock. The methods presented should not only greatly accelerate functional analyses of many aspects of monarch biology, but are also anticipated to facilitate the development of these tools in other nontraditional insect species as well as the development of homology-directed knock-ins.

Molecular identification and characterization of two new Lepidoptera chemoreceptors belonging to the Drosophila melanogaster OR83b family

In insect antennae, olfaction depends on olfactory receptors (ORs) that function through heterodimerization with an unusually highly conserved partner orthologue to the Drosophila melanogaster DOR83b. Here, we report the identification of two cDNAs encoding new DOR83b orthologues that represent the first members, although nonconventional, of the OR families of two noctuid crop pests, the cotton leafworm Spodoptera littoralis and the cabbage armyworm Mamestra brassicae. They both displayed high protein sequence conservation with previously identified DOR83b orthologues. Transcripts were abundantly detected in adult chemosensory organs as well as in fifth instar larvae heads. In adult antennae, the expression patterns of both genes revealed common features with other members of the OR83b subfamily: they appeared to be expressed at the bases of numerous olfactory sensilla belonging to different functional categories, suggesting that both receptors may be co‐expressed with yet unidentified conventional ORs. Bioinformatic analyses predicted the occurrence of seven transmembrane domains and an unusual topology with intracellular N‐termini and extracellular C‐termini, extending to Lepidoptera the hypothesis of an inverted topology for DOR83b orthologues, demonstrated to date only in D. melanogaster.

Molecular Cloning and in Situ Expression Patterns of Two New Pheromone-Binding Proteins from the Corn Stemborer Sesamia nonagrioides

We describe the identification and characterization of two new cDNAs encoding pheromone-binding proteins (PBPs) from the male antennae of Sesamia nonagrioides, a species where no PBPs have been identified to date. Because PBPs are thought to participate in the first step of odor detection in a specific manner, we focused our investigation on this olfactory protein family using reverse transcription–polymerase chain reaction strategies. The deduced amino acid sequences of SnonPBP1 and SnonPBP2 revealed mature proteins of 142 and 143 amino acids, respectively, with six cysteine residues in conserved positions relative to other known PBPs. The alignment of the two mature S. nonagrioides PBPs with other noctuid PBPs showed high sequence identity (70–80%) with other full-length sequences from GenBank. Sequence identity between SnonPBP1 and SnonPBP2 was only 46%, suggesting that the two proteins belong to different classes of PBPs already described from the Noctuidae. Furthermore, analyses of expression patterns of SnonPBP1 and SnonPBP2 were performed by in situ hybridization on antennae of both sexes, and these studies revealed the expression of the two PBPs at the bases of olfactory sensilla (basiconica or trichodea) from both sexes. The possible binding properties of these two new PBPs are discussed according to their homologies with other known PBPs and S. nonagrioides pheromone components.

Unraveling navigational strategies in migratory insects

Long-distance migration is a strategy some animals use to survive a seasonally changing environment. To reach favorable grounds, migratory animals have evolved sophisticated navigational mechanisms that rely on a map and compasses. In migratory insects, the existence of a map sense (sense of position) remains poorly understood, but recent work has provided new insights into the mechanisms some compasses use for maintaining a constant bearing during long-distance navigation. The best-studied directional strategy relies on a time-compensated sun compass, used by diurnal insects, for which neural circuits have begun to be delineated. Yet, a growing body of evidence suggests that migratory insects may also rely on other compasses that use night sky cues or the Earths magnetic field. Those mechanisms are ripe for exploration.