Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core)

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na⁺/K⁺-ATPase, it is unclear how closely related species such as the nonsequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric-pressure scanning microprobe matrix-assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defences might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.     

Reward Tracking and Memory Decay in the Monarch Butterfly,Danaus plexippus L. (Lepidoptera: Nymphalidae)

Due to their long‐distance migration routes and high longevity, monarch butterflies (Danaus plexippus) are likely to benefit from learning how to discriminate and remember suitable feeding resources. In this study, we assessed monarchs’ abilities to track changing nectar sources over time and to retain learned information presented in two conditioning schedules. Non‐preferred (blue and red) and preferred (yellow) artificial flowers were concomitantly offered to monarchs in a three‐phase experiment. In each phase, flowers of only one color contained sucrose solution, while the others contained water. The rewarding color was changed in each phase. Instantaneous observations were made to assess butterfly visits to each color during each phase; continuous observations over the first 90 min of a new phase allowed us to look in more detail at the transition process. Overall, monarchs tracked sucrose availability, visiting the rewarding flowers more often than the unrewarding ones, regardless of innate preferences. However, butterflies reverted to innate color preferences when the newly rewarding color was different from the initial trained color. In a second experiment, memory decay was compared for butterflies trained according to two schedules: ‘single training’ (sucrose solution in red vs. water in blue artificial flowers in one 15‐min session per day) or ‘intermittent training’ (as above, but in two 7.5‐min sessions per day). Afterwards, butterflies were tested on alternate days for a week in arrays containing unrewarding models of both colors. Following either training schedule, memory persisted for at least 3 d after reinforcement ceased. Our findings reveal that monarchs are able to change their feeding responses according to the flowers’ reward status despite innate preferences, as well as to retain flower information for about half a week regardless of the conditioning dynamics.     

The lesser wanderer butterfly, Danaus petilia (Stoll 1790) stat. rev. (Lepidoptera: Danainae), reinstated as a species

Abstract  The lesser wanderer butterfly, Danaus ( Anosia ) chrysippus petilia (Stoll) (Lepidoptera: Danainae), has been treated as a subspecies for the last 100 years. New mitochondrial DNA sequence data for D. petilia , in conjunction with allozyme, structural, morphometric and pattern characters, constitute a compelling case for its specific rank. The holotype of D. petilia has never been located and, as the type location is uncertain, a neotype is designated. Fresh material was collected and examined for this project. Danaus petilia and D. chrysippus have been separated at Lydekker's Line for an estimated 1.1 million years, and they remain interfertile. However, as an allopatric taxon, diagnosable from D. chrysippus , D. petilia merits specific status under the phylogenetic species concept. The following pairs of D. chrysippus subspecies are considered to be synonyms, the first member of each pair having priority: chrysippus  +  aegyptius (subspecies), klugii  +  infumata (hybrid phenotypes) and orientis  + liboria (subspecies).     

Sex differences but no evidence of quantitative honesty in the warning signals of six‐spot burnet moths (Zygaena filipendulae L.)*

The distinctive black and red wing pattern of six‐spot burnet moths (Zygaena filipendulae, L.) is a classic example of aposematism, advertising their potent cyanide‐based defences. While such warning signals provide a qualitatively honest signal of unprofitability, the evidence for quantitative honesty, whereby variation in visual traits could provide accurate estimates of individual toxicity, is more equivocal. Combining measures of cyanogenic glucoside content and wing color from the perspective of avian predators, we investigate the relationship between coloration and defences in Z. filipendulae, to test signal honesty both within and across populations. There were no significant relationships between mean cyanogenic glucoside concentration and metrics of wing coloration across populations in males, yet in females higher cyanogenic glucoside levels were associated with smaller and lighter red forewing markings. Trends within populations were similarly inconsistent with quantitative honesty, and persistent differences between the sexes were apparent: larger females, carrying a greater total cyanogenic glucoside load, displayed larger but less conspicuous markings than smaller males, according to several color metrics. The overall high aversiveness of cyanogenic glucosides and fluctuations in color and toxin levels during an individual's lifetime may contribute to these results, highlighting generally important reasons why signal honesty should not always be expected in aposematic species.     

Contact chemoreception related to host selection and oviposition behaviour in the monarch butterfly, Danaus plexippus

Abstract. Behavioural events during host selection by ovipositing monarch butterflies (Danaus plexippus (L.), Danainae, Nymphalidae) include tapping the leaf surface with fore-tarsi and touching this surface with mid-tarsi (‘drumming’) and antennae. Flavonoids identified from host plant extracts are known to stimulate oviposition. Scanning electron microscopy revealed the presence of contact-chemoreceptor sensilla on all appendages that contact the leaf surface. This electrophysiological study was conducted to identify the contact chemoreceptors that are sensitive to the known oviposition stimuli and are therefore probably involved in host recognition. Receptor cells of conspicuous sensilla grouped in clusters on fore-tarsi of females were sensitive to the behaviourally active butanol fraction of host plant (Asclepias curassavica) extract. However, these receptors generally had low sensitivity to three oviposition-stimulating flavonoids identified from this fraction, but they were also sensitive to the butanol fraction of a non-host (Brassica oleracea). Chemoreceptors in sensilla of the tarsomers 2–4 of the mid-legs also responded to the behaviourally active fraction of host plant extract and showed some sensitivity to two of the flavonoids that stimulate oviposition. Similar results were obtained from receptor cells in sensilla on the tip of the antennae. Most of these sensilla had cells responding to the butanol fraction of A. curassavica extract but only 25% of them were also sensitive to one of the behaviourally active flavonoids. These electrophysiological results, in combination with behavioural observations, suggest that host selection in monarch butterflies relies on a complex pattern of peripheral sensory information from several types of tarsal and antennal contact chemoreceptors.     

Reversal Learning and Risk‐Averse Foraging Behavior in the Monarch Butterfly,Danaus plexippus (Lepidoptera: Nymphalidae)

Learning ability allows insects to respond to a variable environment, and to adjust their behaviors in response to positive or negative experiences. Pollinating insects readily learn to associate floral characteristics, such as color, shape, or pattern, with appetitive stimuli, such as the presence of a nectar reward. However, in nature pollinators may also encounter flowers that contain distasteful or toxic nectar, or offer highly variable nectar volumes, providing opportunities for aversive learning or risk‐averse foraging behavior. Whereas some bees learn to avoid flowers with unpalatable or unreliable nectar rewards, little is known about how Lepidoptera respond to such stimuli. We used a reversal learning paradigm to establish that monarch butterflies learn to discriminate against colored artificial flowers that contain salt solution, decreasing both number of probes and probing time on flowers of a preferred color and altogether avoiding artificial flowers of a non‐preferred color. In addition, when we offered butterflies artificial flowers of two different colors, both of which contained the same mean nectar volume but which differed in variance, the monarchs exhibited risk‐averse foraging: they probed the constant flowers significantly more than the variable ones, regardless of flower color or butterfly sex. Our results add to our understanding of butterfly foraging behavior, as they demonstrate that monarchs can respond to aversive as well as appetitive stimuli, and can also adjust their foraging behavior to avoid floral resources with high variance rewards.     

Little-known accessory glands in female Zygaena moths (Lepidoptera, Zygaenidae)

In the female genital system of Zygaena moths, an additional pair of accessory glands is present besides the Y-shaped sebaceous gland. The term 'Petersen's glands' is proposed for these organs. Anatomy, histology, histochemistry and cytology of Petersen's glands of Zygaena trifolii are described. The sac-like glands, situated in the extreme dorsocaudal part of the abdomen, can be divided into a purely secretory part consisting of acini with large pear-shaped gland cells and a reservoir part with combined secretory and storage function. The secretory cells of the acini are penetrated by long curved ductules or secretory end apparatuses having feltwork consisting of very fine filaments. The cytoplasm is characterized by abundance of smooth tubular endoplasmic reticulum (ATER) and the presence of peroxisomes. This cytoplasmic organization is in accordance with the chemical composition of the sticky secretion, which evidently consists completely of lipids. The ultrastructure of the epithelium lining the reservoir of the glands has both traits of secretory and of transporting epithelia. Besides contributing to the secretion, it may be involved in absorption of residual aqueous phase from the contents of the reservoir.     

Distribution of the monarch butterfly, Danaus plexippus (L.) (Lepidoptera: Nymphalidae), in western North America

The standard model for the migration of the monarch butterfly in western North America has hitherto been movement in the autumn to overwintering sites in coastal California, followed by a return inland by most individuals in the spring. This model is based largely on observational and limited tagging and recovery data. In this paper we test the model by plotting many years of museum and collection records on a monthly basis on a map of the region. Our plots suggest a movement of Oregon, Washington and other north-western populations of summer butterflies to California in the autumn, but movement of more north-easterly populations (e.g. from Idaho and Montana) along two pathways through Nevada, Utah and Arizona to Mexico. The more westerly of these two pathways may follow the Colorado River south as indicated by museum records and seasonal temperature data. The eastern pathway may enter northern Utah along the western scarp of the Wasatch Mountains and run south through Utah and Arizona. Further analysis of distributions suggests that monarch butterflies in the American West occur primarily along rivers, and there are observations indicating that autumn migrants often follow riparian corridors. More data are needed to test our new model; we suggest the nature of the data required.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 85 , 491–500.     

Morphology of the larval and adult brains of the monarch butterfly, Danaus plexippus plexippus, L

Cell population and neuropile morphology of larval and adult brains of the monarch butterfly, Danaus plexippus plexippus, L., are compared. The larval brain is in continuous transition, the processes of adult brain development being underway from the earliest larval stages. It is characterized by a less diverse population of cells and more homogenous fiber areas than those of the adult. Neuroblasts, which divide to form the neurones of the adult brain, occur either in discrete proliferation centers or scattered among the larval ganglion cells. The larval brain contains, in addition to small homogeneous antennal centers and a distinct larval optic center, rapidly developing adult optic centers, corpora pedunculata, and protocerebral bridge. The larval brain lacks a central body. Major differences between larval and adult brains are clearly related to the increased dependence of the adult upon sensory input from the eyes and antennae.     

Heterosis, epistasis and linkage disequilibrium in a wild population of the polymorphic butterfly Danaus chrysippus (L.)

The colour polymorphism of the Danaus chrysippus population at Dar es Salaam, East Africa, is controlled at three major loci, each with two alleles. Two of the loci, one governing ground colour and the other forewing pattern, are closely linked. The third locus, determining hindwing pattern, assorts independently. Thirty-eight broods raised from wild mated pairs, Fl and F2 generations gave 857 offspring of 23 genotypes (out of 27 possible). The forewing length, taken as an index of size, was investigated in relation to the genotype. Heterosis is evident at all three loci. The two linked loci show epistatic interaction of an unexpected kind: double heterozygotes are smaller than heterozygotes at only one locus but larger than double homozygotes. The heterotic effect at the third, unlinked locus is the most pronounced and is additive to that at the other two. Heterosis is more marked in males than females. The possibility that body size has importance in connexion with sexual selection, food resources and mimetic relationships is discussed. Analysis of gene and chromosome frequencies in the wild parents of 61 broods suggests that double heterozygotes for the two linked loci may have heterozygous advantage. Seventy-eight per cent of chromosomes are repulsion phase: thus, there is pronounced linkage disequilibrium which must be maintained by selection as crossing over is almost 296. In particular, the chromosome carrying both dominant alleles in coupling is rare. Consideration of the centres of distribution and present ranges of the alleles at all three loci suggests that three geographical races, aegyptius, dorippus and alcippus, were isolated by forest barriers, during wet periods in the Pleistocene, in south-west, north-east and north-west Africa respectively. They have probably expanded their ranges in the post-glacial period to overlap and interbreed in central and east Africa. Either heterozygous advantage or seasonal (directional) selection or a combination of both is responsible for the persistence of the polymorphism.     

Allozymic and morphometric analysis of populations in the Zygaena purpuralis complex (Lepidoptera, Zygaenidae)

In Central Europe and Asia Minor Zygaena purpuralis and Z. minos are two sibling species exhibiting a considerable geographic variation in several features. In this study we examine some populations from the Central and Southern Apennines (Italy) by comparing multivariate analyses of individual allozymic multilocus profiles and of individual morphometric profiles of male genitalia in order to assess their genetic and phenetic relationships.
Results indicate that populations of Z. purpuralis in the Apennines behave in rather different ways with respect to the occurrence of gene exchange with other gene pools. No gene flow appears to occur between two geographically close southern populations; however, extensive introgression, revealable at both the allozymic and morphometric level, seems to occur between some purpuralis and minos -like sympatric populations from Abruzzi. Evolutionary relationships among these peninsular Italy populations are discussed with reference to historical factors, that is, the occurrence of multiple Pleistocene refugia, which could have determined different degrees of isolation among gene pools. This situation is rather different from that previously outlined for Central Europe and present results suggest that the Z. purpuralis complex might include more than two biological species.     

Additive genetic variation,but not temperature,influences warning signal expression in Amata nigriceps moths (Lepidoptera: Arctiinae)

Many aposematic species show variation in their color patterns even though selection by predators is expected to stabilize warning signals toward a common phenotype. Warning signal variability can be explained by trade‐offs with other functions of coloration, such as thermoregulation, that may constrain warning signal expression by favoring darker individuals. Here, we investigated the effect of temperature on warning signal expression in aposematic Amata nigriceps moths that vary in their black and orange wing patterns. We sampled moths from two flight seasons that differed in the environmental temperatures and also reared different families under controlled conditions at three different temperatures. Against our prediction that lower developmental temperatures would reduce the warning signal size of the adult moths, we found no effect of temperature on warning signal expression in either wild or laboratory‐reared moths. Instead, we found sex‐ and population‐level differences in wing patterns. Our rearing experiment indicated that ~70% of the variability in the trait is genetic but understanding what signaling and non‐signaling functions of wing coloration maintain the genetic variation requires further work. Our results emphasize the importance of considering both genetic and plastic components of warning signal expression when studying intraspecific variation in aposematic species.     

Spring remigration of the monarch butterfly, Danaus plexippus (Lepidoptera: Nymphalidae) in north-central Florida: estimating population parameters using mark-recapture

Monarch butterflies [Danaus plexippus) of the eastern North American population migrate each fall from die northern U.S.A. and southern Canada to overwintering sites in Mexico and return the following spring to the southeastern U.S.A. where they lay eggs and then die. The spring remigration is the least studied phase in the annual migration cycle. We therefore conducted a mark-recapture study and examined population recolonization dynamics and residence time in a north-central Florida pasture where the monarch's milkweed host plant (Asclepias humistrata) was abundant. Beginning in late March 1995 two waves of monarchs arrived, their numbers peaked at 71 individuals by mid-April, and the butterflies disappeared in early May. After arriving, the adults remained for 3–5 days, laid eggs and then continued to migrate. We also compared population sizes and arrival times in 1994 and 1996. We found no evidence of a second spring generation, which was also consistent with the deteriorating quality of the A. humistrata plants. Individuals of the new spring generation disappear shordy after eclosion. The arriving population was approximately nine times greater in 1995 than in 1996. Our findings support two recent hypodieses: (1) the bird-like migration of the monarch butterfly in North America evolved with the northward expansion and phenology of milkweeds; and (2) monarchs appear to be migratory throughout their annual cycle of several generations. By lingering for only a short time at each milkweed patch they encounter, the old monarchs returning from Mexico locate the resurgent milkweed flora over an extensive area in the southern states. Then, within less than a month, their fresh offspring continue the migration and exploit the unfolding cornucopia of milkweeds as the spring advances northward. The more we discover about the biology of this insect, the more remarkable is its annual migratory, breeding and overwintering cycle.     

Pre- and postzygotic isolation and Haldane rule effects in reciprocal crosses of Danaus erippus and Danaus plexippus (Lepidoptera: Danainae), supported by differentiation of cuticular hydrocarbons, establish their status as separate species

Hybridization experiments and cuticular hydrocarbon analyses were performed to determine the extent of the reproductive isolation present to clarify the taxonomic status of Danaus erippus (Cramer, 1775), which has often been classified as a subspecies of Danaus plexippus (Linnaeus, 1758) . The results of crosses between D. erippus and Danaus plexippus nigrippus (Haensch, 1909) showed pre- and postzygotic isolation. Pupal inviability, and Haldane rule effects (male hybrids only, the homogametic sex in butterflies) were observed after the crosses. These results reinforce the hypothesis that D. plexippus and D. erippus are separate, reproductively isolated species. However, we have also investigated the cuticular hydrocarbons in D. erippus and D. p. nigrippus with temperature-programmed gas chromatography. The chromatographic profiles of peaks were similar in both taxa, with retention indices in the range 2100–3797. The profiles were characterized by the presence of 15 peaks whose retention indices were in the range 2501–3772. Although no major qualitative differences between the two species were observed, we did find some quantitative differences. A principal component analysis showed a clear separation between members of D. erippus and D. p. nigrippus , suggesting that semiochemical differences might have become differentiated after isolation, perhaps in response to different physiological pressures.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 445–453.     

Experimental Examination of Intraspecific Density-Dependent Competition during the Breeding Period in Monarch Butterflies (Danaus plexippus)

A central goal of population ecology is to identify the factors that regulate population growth. Monarch butterflies (Danaus plexippus) in eastern North America re-colonize the breeding range over several generations that result in population densities that vary across space and time during the breeding season. We used laboratory experiments to measure the strength of density-dependent intraspecific competition on egg laying rate and larval survival and then applied our results to density estimates of wild monarch populations to model the strength of density dependence during the breeding season. Egg laying rates did not change with density but larvae at high densities were smaller, had lower survival, and weighed less as adults compared to lower densities. Using mean larval densities from field surveys resulted in conservative estimates of density-dependent population reduction that varied between breeding regions and different phases of the breeding season. Our results suggest the highest levels of population reduction due to density-dependent intraspecific competition occur early in the breeding season in the southern portion of the breeding range. However, we also found that the strength of density dependence could be almost five times higher depending on how many life-stages were used as part of field estimates. Our study is the first to link experimental results of a density-dependent reduction in vital rates to observed monarch densities in the wild and show that the effects of density dependent competition in monarchs varies across space and time, providing valuable information for developing robust, year-round population models in this migratory organism.     

The structure and function of the bursa copulatrix of the monarch butterfly (Danaus plexippus)

The bursa compulatrix of the Monarch butterfly was investigated utilizing light microscopy, histochemistry, and scanning and transmission electron microscopy in order to relate its morphology to the release of sperm from the spermatophore. The bursa has a row of large chitinous teeth on either side of the organ. The dorsal and ventral surfaces are covered with chitinous plates, the plates having bristles on one side. A single layer of cells lies under both the plates and teeth, one columnar cell under each plate, one cuboidal cell under each tooth. The toothed area has no muscle cells. However, the dorsal and ventral hemispheres of the bursa each have a crescent-shaped packet of muscle fibers that traverse the organ; there are no longitudinal fibers. Spermatophores with thick walls were found in the bursal lumen. Morphological evidence suggests that the presence of the spermatophores is sensed by the bristles and that the packets are opened by contraction of the muscles bringing the large teeth into contact with the spermatophore wall.     

Thoracic temperature,shivering, and flight in the monarch butterfly,Danaus plexippus (L.)

Summary Monarch butterflies, Danaus plexippus (L.), display a warm-up behavior characterized by wingstrokes of small amplitude. Thoracic temperature during this shivering and during fixed flight was measured by means of a smallbead thermistor inserted into the thorax. At ambient temperatures of 15–16°C, once shivering is initiated the thoracic temperature rises at a maximum rate of 1.3°C/min, and a thoracic temperature 4.0°C greater then ambient is produced (Table 1). Fixed flight at these low ambient temperatures results in a similar rate of increase in thoracic temperature, and a similar temperature excess is produced (Fig. 3). At ambient temperatures between 22 and 35°C the thoracic temperature of an animal starting to fly rises at a faster rate, 3.6°C/min, and reaches a greater excess, 7.9°C (Fig. 4). The wingbeat frequency of animals in fixed flight increases with increasing thoracic temperature (Fig. 2). In the absence of direct solar radiation, shivering typically occurs prior to flight at low ambient temperatures (13–17°C), and the resulting increase in thoracic temperature allows monarch butterflies to fly at these cool temperatures.I thank Miss Janice Ruppert and Mr. C. J. Doughty for their valuable technical assistance. The co-operation of the administrators of New Brighton Beach State Park in permitting me to collect in the park is appreciated. Financial support for this study was provided in part by a faculty research grant from the University of California.     

Dynamics of oviposition in Danaus plexippus (Insecta: Lepidoptera) on milkweed, Asclepias spp

Where a butterfly chooses to lay an egg will influence the subsequent survival of her offspring. In this paper we report on the effect of a number of variables which influence the choice of oviposition site in Danaus plexippus L. Experiments were conducted under both flight cage and field conditions. The field observations consisted of recording the within and between plant egg dispersions across different patch sizes. Laboratory experiments looked at egg laying preferences as affected by plant species, age and condition. Butterflies were selective in their choice of oviposition site. Eggs were laid singly on the underside of medium sized leaves towards the top of a plant. More eggs were laid per plant on single isolated plants than on plants within a patch. The number of eggs per plant increases with plant height but decreases with plant age. Females preferred young plants or plants with fresh regrowth of leaves. These characteristics could override species preferences which were, in decreasing order, Asclepias curassavica. A. fruticosa and A. physocarpa. Species preferences varied between butterflies and with female age. Neither the presence of eggs nor larvae on a plant deterred oviposition. These results are compared with previous observations of egg laying in this species.     

The Effect of Behavior and Ecology on Male Mating Success in Overwintering Monarch Butterflies (Danaus plexippus)

Monarch butterflies (Danaus plexippus) appear to forego the chemical courtship that is typical of other danaid butterflies, and instead employ a coercive mating system. Females have been described as using resistance behaviors in response to male coercion. Much of our understanding of sexual selection in monarchs is based on observations of mating attempts that occur on the ground, but recent studies report frequent mating attempts in the tree canopy. I compared mating activity on the ground to that in the tree canopy and found that, among heterosexual mating attempts, location did not affect the likelihood of ending in copulation. This suggests that the more easily obtained data on ground attempts may reasonably approximate mating activity in the canopy. The outcome of ground attempts was influenced by vegetation structure and the position of the male relative to the female and the ground at the start of the ground phase of the mating attempt. My observations suggest that butterfly position may be correlated with male search strategy. It remains unclear whether described resistance behaviors represent female or male control. However, the data do demonstrate that attempt outcome is influenced by both behavior and ecology: male search and capture strategies may influence copulatory success, and human-induced changes to the habitat can influence monarch mating activity at overwintering sites.