3D reconstruction of larval and adult brain neuropils of two giant silk moth species: Hyalophora cecropia and Antheraea pernyi.

We present a detailed analysis of the brain anatomy of two saturniid species, the cecropia silk moth, Hyalophora cecropia, and the Chinese oak silk moth, Antheraea pernyi, including 3D reconstructions of the major brain neuropils in the larva and in male and female adults. The 3D reconstructions, prepared from high-resolution optical sections, showed that the corresponding neuropils of these saturniid species are virtually identical. Similarities between the two species include a pronounced sexual dimorphism in the adults in the form of a male-specific assembly of markedly enlarged glomeruli forming the so-called macroglomerular complex. From the reports published to date, it can be concluded that the neuropil architecture of saturniids resembles that of other nocturnal moths, including the sibling family Sphingidae. In addition, compared with previous anatomical data on diurnal lepidopteran species, significant differences were observed in the two saturniid species, which include the thickness of the Y-tract of the mushroom body, the size of the main neuropils of the optic lobes, and the sexual dimorphisms of the antennal lobes.

A re-evaluation of silk measurement by the cecropia caterpillar (Hyalophora cecropia) during cocoon construction reveals use of a silk odometer that is temporally regulated.

The late 5th instar caterpillar of the cecropia silk moth (Hyalophora cecropia) spins a silken cocoon with a distinct, multilayered architecture. The cocoon construction program, first described by the seminal work of Van der Kloot and Williams, consists of a highly ordered sequence of events. We perform behavioral experiments to re-evaluate the original cecropia work, which hypothesized that the length of silk that passes through the spinneret controls the orderly execution of each of the discrete events of cocoon spinning. We confirm and extend by three-dimensional scanning and quantitative measurements of silk weights that if cocoon construction is interrupted, upon re-spinning, the caterpillar continues the cocoon program from where it left off. We also confirm and extend by quantitative measurements of silk weights that cecropia caterpillars will not bypass any of the sections of the cocoon during the construction process, even if presented with a pre-spun section of a cocoon spun by another caterpillar. Blocking silk output inhibits caterpillars from performing normal spinning behaviors used for cocoon construction. Surprisingly, unblocking silk output 24-hr later did not restart the cocoon construction program, suggesting the involvement of a temporally-defined interval timer. We confirm with surgical reductions of the silk glands that it is the length of silk itself that matters, rather than the total amount of silk extracted by individuals. We used scanning electron microscopy to directly show that either mono- or dual-filament silk (i.e., equal silk lengths but which vary in their total amount of silk extracted) can be used to construct equivalent cocoons of normal size and that contain the relevant layers. We propose that our findings, taken together with the results of prior studies, strongly support the hypothesis that the caterpillar uses a silk "odometer" to measure the length of silk extracted during cocoon construction but does so in a temporally regulated manner. We further postulate that our examination of the anatomy of the silk spinning apparatus and ablating spinneret sensory output provides evidence that silk length measurement occurs upstream of output from the spinneret.

Demystifying Monarch Butterfly Migration.

Every fall, millions of North American monarch butterflies undergo a stunning long-distance migration to reach their overwintering grounds in Mexico. Migration allows the butterflies to escape freezing temperatures and dying host plants, and reduces infections with a virulent parasite. We discuss the multigenerational migration journey and its evolutionary history, and highlight the navigational mechanisms of migratory monarchs. Monarchs use a bidirectional time-compensated sun compass for orientation, which is based on a time-compensating circadian clock that resides in the antennae, and which has a distinctive molecular mechanism. Migrants can also use a light-dependent inclination magnetic compass for orientation under overcast conditions. Additional environmental features, e.g., atmospheric conditions, geologic barriers, and social interactions, likely augment navigation. The publication of the monarch genome and the development of gene-editing strategies have enabled the dissection of the genetic and neurobiological basis of the migration. The monarch butterfly has emerged as an excellent system to study the ecological, neural, and genetic basis of long-distance animal migration.

Dimorphic cocoons of the cecropia moth (Hyalophora cecropia): Morphological, behavioral, and biophysical differences.

The larvae of the giant silk moth (Hyalophora cecropia) spin strikingly dimorphic, multilayered cocoons that are either large and fluffy (baggy) or significantly smaller and tightly woven (compact). Although these cocoon-morphs share the same function (i.e., housing for pupal to adult development during overwintering), previous work has been unable to determine why cocoon dimorphism exists. We addressed this issue in cecropia moth cocoons collected along power line right-of-way habitats in Massachusetts. We first characterized the architectural differences between cocoon-morphs for all three cocoon sections (outer and inner envelopes, and the intermediate layer separating the two). We show that outer envelope structural and ultrastructural differences are what underlie dimorphism. Using a common spinning arena, we next show that the behavioral suites used to construct the outer envelopes of the two morphs are significantly different in behavioral time investment and patterning, as well as in the location of silk placement in the common spinning arena. Finally, we compared the cocoon-morphs in response to various environmental stressors to ask whether dimorphism is an adaptive response to such pressures. In contrast to compact cocoons, we find that baggy cocoons act as heat sinks and allow greater moisture permeability; differences in outer envelope architecture underlie these characteristics. These two biophysical properties could be advantageous for pupae in baggy cocoons, during unseasonably cold or dry conditions encountered during development prior to adult emergence. Our results suggest that cocoon dimorphism in the cecropia moth may provide a bet-hedging strategy for dealing with varying environmental conditions in Massachusetts and perhaps over its entire habitat range, during pupal to adult development.

Neural Integration Underlying a Time-Compensated Sun Compass in the Migratory Monarch Butterfly.

Migrating eastern North American monarch butterflies use a time-compensated sun compass to adjust their flight to the southwest direction. Although the antennal genetic circadian clock and the azimuth of the sun are instrumental for proper function of the compass, it is unclear how these signals are represented on a neuronal level and how they are integrated to produce flight control. To address these questions, we constructed a receptive field model of the compound eye that encodes the solar azimuth. We then derived a neural circuit model that integrates azimuthal and circadian signals to correct flight direction. The model demonstrates an integration mechanism, which produces robust trajectories reaching the southwest regardless of the time of day and includes a configuration for remigration. Comparison of model simulations with flight trajectories of butterflies in a flight simulator shows analogous behaviors and affirms the prediction that midday is the optimal time for migratory flight.

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.

Neurobiology of Monarch Butterfly Migration.

Studies of the migration of the eastern North American monarch butterfly (Danaus plexippus) have revealed mechanisms behind its navigation. The main orientation mechanism uses a time-compensated sun compass during both the migration south and the remigration north. Daylight cues, such as the sun itself and polarized light, are processed through both eyes and integrated through intricate circuitry in the brain's central complex, the presumed site of the sun compass. Monarch circadian clocks have a distinct molecular mechanism, and those that reside in the antennae provide time compensation. Recent evidence shows that migrants can also use a light-dependent inclination magnetic compass for orientation in the absence of directional daylight cues. The monarch genome has been sequenced, and genetic strategies using nuclease-based technologies have been developed to edit specific genes. The monarch butterfly has emerged as a model system to study the neural, molecular, and genetic basis of long-distance animal migration.

Sensory basis of lepidopteran migration: focus on the monarch butterfly.

In response to seasonal habitats, migratory lepidopterans, exemplified by the monarch butterfly, have evolved migration to deal with dynamic conditions. During migration, monarchs use orientation mechanisms, exploiting a time-compensated sun compass and a light-sensitive inclination magnetic compass to facilitate fall migration south. The sun compass is bidirectional with overwintering coldness triggering the change in orientation direction for remigration northward in the spring. The timing of the remigration and milkweed emergence in the southern US have co-evolved for propagation of the migration. Current research is uncovering the anatomical and molecular substrates that underlie migratory-relevant sensory mechanisms with the antennae being critical components. Orientation mechanisms may be detrimentally affected by environmental factors such as climate change and sensory interference from human-generated sources.

The genetics of monarch butterfly migration and warning colouration.

The monarch butterfly, Danaus plexippus, is famous for its spectacular annual migration across North America, recent worldwide dispersal, and orange warning colouration. Despite decades of study and broad public interest, we know little about the genetic basis of these hallmark traits. Here we uncover the history of the monarch's evolutionary origin and global dispersal, characterize the genes and pathways associated with migratory behaviour, and identify the discrete genetic basis of warning colouration by sequencing 101 Danaus genomes from around the globe. The results rewrite our understanding of this classic system, showing that D. plexippus was ancestrally migratory and dispersed out of North America to occupy its broad distribution. We find the strongest signatures of selection associated with migration centre on flight muscle function, resulting in greater flight efficiency among migratory monarchs, and that variation in monarch warning colouration is controlled by a single myosin gene not previously implicated in insect pigmentation.

A magnetic compass aids monarch butterfly migration.

Convincing evidence that migrant monarch butterflies (Danaus plexippus) use a magnetic compass to aid their fall migration has been lacking from the spectacular navigational capabilities of this species. Here we use flight simulator studies to show that migrants indeed possess an inclination magnetic compass to help direct their flight equatorward in the fall. The use of this inclination compass is light-dependent utilizing ultraviolet-A/blue light between 380 and 420 nm. Notably, the significance of light <420 nm for inclination compass function was not considered in previous monarch studies. The antennae are important for the inclination compass because they appear to contain light-sensitive magnetosensors. For migratory monarchs, the inclination compass may serve as an important orientation mechanism when directional daylight cues are unavailable and may also augment time-compensated sun compass orientation for appropriate directionality throughout the migration.

Coldness triggers northward flight in remigrant monarch butterflies.

Each fall, eastern North American monarch butterflies (Danaus plexippus) migrate from their northern range to their overwintering grounds in central Mexico. Fall migrants are in reproductive diapause, and they use a time-compensated sun compass to navigate during the long journey south. Eye-sensed directional cues from the daylight sky (e.g., the horizontal or azimuthal position of the sun) are integrated in the sun compass in the midbrain central complex region. Sun compass output is time compensated by circadian clocks in the antennae so that fall migrants can maintain a fixed flight direction south. In the spring, the same migrants remigrate northward to the southern United States to initiate the northern leg of the migration cycle. Here we show that spring remigrants also use an antenna-dependent time-compensated sun compass to direct their northward flight. Remarkably, fall migrants prematurely exposed to overwintering-like coldness reverse their flight orientation to the north. The temperature microenvironment at the overwintering site is essential for successful completion of the migration cycle, because without cold exposure, aged migrants continue to orient south. Our discovery that coldness triggers the northward flight direction in spring remigrants solves one of the long-standing mysteries of the monarch migration.

Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases.

The development of reverse-genetic tools in "nonmodel" insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into "one nucleus" stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and "nonmodel" insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes.

Anatomical basis of sun compass navigation II: the neuronal composition of the central complex of the monarch butterfly.

Each fall, eastern North American monarch butterflies in their northern range undergo a long-distance migration south to their overwintering grounds in Mexico. Migrants use a time-compensated sun compass to determine directionality during the migration. This compass system uses information extracted from sun-derived skylight cues that is compensated for time of day and ultimately transformed into the appropriate motor commands. The central complex (CX) is likely the site of the actual sun compass, because neurons in this brain region are tuned to specific skylight cues. To help illuminate the neural basis of sun compass navigation, we examined the neuronal composition of the CX and its associated brain regions. We generated a standardized version of the sun compass neuropils, providing reference volumes, as well as a common frame of reference for the registration of neuron morphologies. Volumetric comparisons between migratory and nonmigratory monarchs substantiated the proposed involvement of the CX and related brain areas in migratory behavior. Through registration of more than 55 neurons of 34 cell types, we were able to delineate the major input pathways to the CX, output pathways, and intrinsic neurons. Comparison of these neural elements with those of other species, especially the desert locust, revealed a surprising degree of conservation. From these interspecies data, we have established key components of a conserved core network of the CX, likely complemented by species-specific neurons, which together may comprise the neural substrates underlying the computations performed by the CX.

MonarchBase: the monarch butterfly genome database.

The monarch butterfly (Danaus plexippus) is emerging as a model organism to study the mechanisms of circadian clocks and animal navigation, and the genetic underpinnings of long-distance migration. The initial assembly of the monarch genome was released in 2011, and the biological interpretation of the genome focused on the butterfly's migration biology. To make the extensive data associated with the genome accessible to the general biological and lepidopteran communities, we established MonarchBase (available at http://monarchbase.umassmed.edu). The database is an open-access, web-available portal that integrates all available data associated with the monarch butterfly genome. Moreover, MonarchBase provides access to an updated version of genome assembly (v3) upon which all data integration is based. These include genes with systematic annotation, as well as other molecular resources, such as brain expressed sequence tags, migration expression profiles and microRNAs. MonarchBase utilizes a variety of retrieving methods to access data conveniently and for integrating biological interpretations.

Discordant timing between antennae disrupts sun compass orientation in migratory monarch butterflies.

To navigate during their long-distance migration, monarch butterflies (Danaus plexippus) use a time-compensated sun compass. The sun compass timing elements reside in light-entrained circadian clocks in the antennae. Here we show that either antenna is sufficient for proper time compensation. However, migrants with either antenna painted black (to block light entrainment) and the other painted clear (to permit light entrainment) display disoriented group flight. Remarkably, when the black-painted antenna is removed, re-flown migrants with a single, clear-painted antenna exhibit proper orientation behaviour. Molecular correlates of clock function reveal that period and timeless expression is highly rhythmic in brains and clear-painted antennae, while rhythmic clock gene expression is disrupted in black-painted antennae. Our work shows that clock outputs from each antenna are processed and integrated together in the monarch time-compensated sun compass circuit. This dual timing system is a novel example of the regulation of a brain-driven behaviour by paired organs.

Anatomical basis of sun compass navigation I: the general layout of the monarch butterfly brain.

Each fall, eastern North American monarch butterflies (Danaus plexippus) use a time-compensated sun compass to migrate to their overwintering grounds in central Mexico. The sun compass mechanism involves the neural integration of skylight cues with timing information from circadian clocks to maintain a constant heading. The neuronal substrates for the necessary interactions between compass neurons in the central complex, a prominent structure of the central brain, and circadian clocks are largely unknown. To begin to unravel these neural substrates, we performed 3D reconstructions of all neuropils of the monarch brain based on anti-synapsin labeling. Our work characterizes 21 well-defined neuropils (19 paired, 2 unpaired), as well as all synaptic regions between the more classically defined neuropils. We also studied the internal organization of all major neuropils on brain sections, using immunocytochemical stainings against synapsin, serotonin, and γ-aminobutyric acid. Special emphasis was placed on describing the neuroarchitecture of sun-compass-related brain regions and outlining their homologies to other migratory species. In addition to finding many general anatomical similarities to other insects, interspecies comparison also revealed several features that appear unique to the monarch brain. These distinctive features were especially apparent in the visual system and the mushroom body. Overall, we provide a comprehensive analysis of the brain anatomy of the monarch butterfly that will ultimately aid our understanding of the neuronal processes governing animal migration.

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 Earth's magnetic field. Those mechanisms are ripe for exploration.

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.

Human cryptochrome exhibits light-dependent magnetosensitivity.

Humans are not believed to have a magnetic sense, even though many animals use the Earth's magnetic field for orientation and navigation. One model of magnetosensing in animals proposes that geomagnetic fields are perceived by light-sensitive chemical reactions involving the flavoprotein cryptochrome (CRY). Here we show using a transgenic approach that human CRY2, which is heavily expressed in the retina, can function as a magnetosensor in the magnetoreception system of Drosophila and that it does so in a light-dependent manner. The results show that human CRY2 has the molecular capability to function as a light-sensitive magnetosensor and reopen an area of sensory biology that is ready for further exploration in humans.