Monarch Butterfly Migration Moving into the Genetic Era

Abstract

The genetic architecture and neurogenetics of animal migration remain poorly understood. With a sequenced genome and the establishment of reverse genetic tools, the monarch butterfly has emerged as a promising model to uncover the genetic basis of migratory behavior and associated traits. Here, we synthesize major advances made in the genetics of monarch migration, which includes the discovery of genomic regions associated with migration and molecular mechanisms underpinning its seasonality. We highlight the catalytic role that a rapidly growing number of contemporary genetic and molecular technologies applicable to nonconventional organisms have had in these discoveries, and outline new avenues of investigation to continue moving the field forward.

Keywords: Danaus plexippus; functional genomics; genetic tools; migratory behavior.

Figure 1:. Annual migratory cycle of North American monarch butterflies.

North American monarchs start migrating southward in the fall, coincidentally with decreasing daylength sensed by an endogenous timer functioning with circadian clock genes. Monarchs east of the Rocky Mountains (grey line) navigate over long-distances (red arrows) to their overwintering sites in Mexico (blue circle). In the spring, when temperatures and photoperiod increase, the same individuals become reproductive, mate, and reverse their flight orientation northward. The switch in compass orientation in seasonal migratory forms has been shown to result from prolonged exposure to coldness that mimic those experienced at the overwintering sites, underscoring the critical importance to increase conservation efforts of the overwintering sites that are threatened by logging. On their way back to the United States, fertilized remigrant females lay their eggs on milkweed plants before dying (red arrows). Subsequent generations of spring and summer butterflies progress northward following the latitudinal emergence of their host plants to repopulate the northern summer breeding grounds (black arrows). Fall migration, and spring and summer breeding ranges are denoted by colored areas. Monarchs west of the Rockies also migrate southward in the fall, overwinter along the Californian coast (blue line), and remigrate northward in the spring, but the migration distances are much shorter than those travelled by eastern North American monarchs. Modified from [3].

Figure 2:. Genetic dissection of monarch migration using population genomics.

(A) Monarch populations that differ in their migratory phenotypes are distributed around the globe. In North America, two migratory populations separated by the Rocky Mountains undergo seasonal migrations: the Eastern population, best known for its spectacular long-distance migration, overwinters in central Mexico; and the Western population, which migrates over much shorter distances, overwinters on the California coast. A third migratory population is present in Australia. However, monarchs also exist throughout Central America, South America, the Caribbean, Europe, North Africa, and throughout the Pacific islands, where they appear to have formed non-migratory populations through three dispersal events from the ancestral Eastern North American migratory population. Whether they lack the ability to migrate or simply do not express this behavior in their local environments remains an open question. (B) The variation in migratory phenotypes across populations has been leveraged for comparative population genomic studies. Regions of the genome strongly differentiating North American monarchs and monarchs from non-migratory populations were identified by re-sequencing the genome of these individuals and applying quantitative measures of sequence differentiation such as population-branch statistics (PBS). The most highly differentiated region contained three genes encoding the F-box protein FBXO45, an uncharacterized transmembrane protein (DPOGS206536) and the α−1 subunit of collagen type IV. Modified from [6]. (C) Because of their strong association with a shift in migratory behavior, these genes (together with those found in other differentiated genomic regions) may underpin the genetic basis of monarch migration.

Figure 3:. Circadian clocks and the induction of seasonal reproductive diapause.

(A) The core molecular mechanism of the monarch circadian clock relies on a feedback loop in which the CLOCK (CLK) and BMAL1 heterodimer drives the rhythmic transcription of the cryptochrome 2 (cry2), period (per), and timeless (tim) genes. CRY2, PER and TIM form complexes in the cytosol. Upon PER phosphorylation, PER and CRY2 are translocated into the nucleus and repress CLK:BMAL1-mediated transcription. The blue-light circadian photoreceptor CRYPTOCHROME 1 (CRY1) resets the clock by mediating TIM degradation upon light exposure. (B) Expression profiles of cry2, per and tim mRNA levels over a 24-hour day. (C) The circadian clock or clock genes in the brain are involved in the induction of reproductive diapause exhibited by fall migrants. The brain clock helps monarchs distinguish long photoperiods (LP) in the summer from short photoperiods (SP) in the fall. The brain clock affects photoperiodic responsiveness by regulating, in a photoperiod-dependent fashion, the expression of genes involved in the vitamin A pathway. Beta-carotene is transported into extraretinal neural cells of the adult brain via SANTA MARIA, and converted to retinal by the rate-limiting enzyme NINA B. Retinal can either be interconverted into retinol by a retinol dehydrogenase (RDH) or converted into retinoic acid (RA) by a retinaldehyde dehydrogenase (RALDH). RA binds to retinoid receptors to regulate transcription of target genes. Functional disruption of the clock and of the vitamin pathway disrupts photoperiod responsiveness. The connection between vitamin A and juvenile hormone deficiency, characteristic of diapausing monarchs, remains unknown. Modified from [48].

Figure 4:. Integration of timing and sun compass information for flight orientation.

(A) Migrant monarchs housed in fall light:dark (LD) cycles with lights on at 7:00AM and lights off at 7:00PM and flown in a flight simulator in the morning orient in the proper southwesterly migratory direction (upper left). When housed in clock-shifted LD cycles advanced by six hours, monarchs interpret this morning sun as an afternoon sun and shift their orientation counterclockwise, demonstrating time-compensation of sun compass orientation (lower left). Modified from [2]. In contrast to fall migrants with intact antennae, antennae-less migrants are disoriented as a group, showing that the antennae contain the timer for sun compass orientation (upper and lower right). Modified from [18]. Colored dots, orientation of individuals; arrow, mean orientation of the group. (B) Skylight cues are sensed by the eyes (ultraviolet polarized light by the dorsal rim, and colors of the light or the sun itself by the main retina) and integrated in the central complex (CX; blue). Circadian clocks in the antennae provide the major timing information for sun compass orientation behavior, but brain clocks could have a minor contribution. The neural pathways connecting circadian clocks to the CX remain to be determined (red lines with question marks). Ultimately, the integrated signal is transmitted via descending neurons (grey line) to motor circuits to generate oriented flight behavior. Modified from [88]. (C) Genetic tools for genomic integration, including transposon-based transgenesis and CRISPR/Cas9-mediated homology directed repair (HDR), could be employed to mark clock neurons with fluorescent proteins and map the clock circuitry.

Figure 5:. ldness-induced reprogramming of seasonal flight orientation to study the molecular basis of sun compass orientation.

Co (A) Migrants orient southwesterly in the fall, and reverse their flight northeasterly in the spring after prolonged exposure to overwintering coldness conditions (left). Fall migrants subjected to simulated overwintering-like coldness for 24 days in constant photoperiod also reverse their flight orientation northward (right). Modified from [16]. (B) The switch in flight orientation upon exposure to environmental coldness suggests an epigenetic reprogramming of flight orientation. The genes, cis-regulatory elements (CREs) and putative transcription factors (TFs) that control their expression, which may be involved in this molecular switch, could be identified through integrated approaches combining RNA-seq, ATAC-seq and CUT&RUN in brains of fall migrants and cold-treated fall migrants. RNA-seq quantifies differential gene expression between conditions. ATAC-seq detects open chromatin regions as well as TF footprints for the identification of putative TFs. CUT&RUN profiles the epigenome through the use of antibodies against conserved histone marks enriched in permissive or repressive chromatin regions.