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Identification of cytochrome P450 and glutathione-S-transferase genes preferentially expressed in chemosensory organs of the swallowtail butterfly, Papilio xuthus L.

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. We undertook an expressed sequence tag (EST) analysis to identify the chemosensory-related genes that are expressed in chemosensilla on the tarsus of P. xuthus. Several genes that showed similarity with biotransformation enzymes were identified from the ESTs. Among them, a cytochrome P450 and a glutathione-S-transferase (GST) were preferentially expressed in the chemosensory organs. We have determined the structure of both cDNA and genomic sequences encoding these enzymes and designated the P450 as CYP341A2, a novel member of CYP341A subfamily, and the GST as GST-pxcs1, respectively. We observed a localized expression of CYP341A2 at the base of tarsal chemosensilla by in situ hybridization. These results suggest that these degrading enzymes play a role in the chemosensory reception for host plant recognition.

Amino Acid Sequence↗

Effects of adult-derived carbohydrates, amino acids and micronutrients on female reproduction in a fruit-feeding butterfly.

It is generally believed that butterflies (and other holometabolous insects) rely primarily on reserves accumulated during the larval stage for reproduction, whereas the carbohydrate-rich adult diet is thought to mainly cover energy requirements. In at least some species though, realization of the full reproductive potential is extensively affected by post-eclosion nutrition. While the importance of carbohydrates is fairly well understood, the role of adult-derived amino acids and micronutrients is controversial and largely unknown, respectively. We here focus on the effects of different adult diets on female reproduction in the tropical, fruit-feeding butterfly Bicyclus anynana (Nymphalidae). Carbohydrates were the most important adult-derived nutrients affecting reproduction. Adding amino acids, vitamins or minerals to sucrose-based solutions did not yield a reproductive output equivalent to that of fruit-fed females, which showed the highest performance throughout. This suggests that either not yet identified compounds of fruit substantially contribute to reproduction, or that resource congruence (the use of nutrient types in a specified ratio) rather than any specific nutrient component is of key importance. Apart from adult income, realized fecundity depended on egg size and longevity, with the former dominating when dietary quality was low, but the latter when quality was high. Thus, the egg size-number trade-off seems to be affected by female nutrition.

Amino Acids↗

Connecting the navigational clock to sun compass input in monarch butterfly brain.

Migratory monarch butterflies (Danaus plexippus) use a time-compensated sun compass to navigate to their overwintering grounds in Mexico. Although polarized light is one of the celestial cues used for orientation, the spectral content (color) of that light has not been fully explored. We cloned the cDNAs of three visual pigment-encoding opsins (ultraviolet [UV], blue, and long wavelength) and found that all three are expressed uniformly in main retina. The photoreceptors of the polarization-specialized dorsal rim area, on the other hand, are monochromatic for the UV opsin. Behavioral studies support the importance of polarized UV light for flight orientation. Next, we used clock protein expression patterns to identify the location of a circadian clock in the dorsolateral protocerebrum of butterfly brain. To provide a link between the clock and the sun compass, we identified a CRYPTOCHROME-staining neural pathway that likely connects the circadian clock to polarized light input entering brain.

Animal Migration↗

Pharmacologic approaches to butterfly wing patterning: sulfated polysaccharides mimic or antagonize cold shock and alter the interpretation of gradients of positional information.

Butterflies produce complex and diverse wing patterns by mechanisms that are generally unknown. We have employed a pharmacological approach to explore the molecular mechanisms of pattern formation. In a screen of over 200 compounds injected into developing Junonia coenia pupae, we identified several specific sulfated polysaccharides that caused widespread, dose-dependent effects on adult wing patterns. These compounds were well tolerated and permitted butterflies to eclose normally and take flight at moderate levels of effect. Heparin and closely related chondroitin sulfates caused stage-specific expansion of distal and proximal band systems and reduction and repatterning of eyespots. Dextran sulfate and fucoidan, whose structures are widely divergent from heparin and one another, caused contraction of distal and proximal systems, but had no effect on eyespots. Nonsulfated or nonpolymeric saccharides were without effect. Pattern alterations were indistinguishable from those reported for extreme cold shock and exposure to sodium tungstate and "molsin". When administered after cold shock or coinjected with heparin, dextran sulfate reversed all patterning effects. We suggest that the primary effect of polysaccharide treatments is to alter the interpretation of gradients of positional information along the proximodistal axis of the pupal wing.

Animals↗

The importance of time scale and multiple refugia: incipient speciation and admixture of lineages in the butterfly Erebia triaria (Nymphalidae).

Evidence of four different Quaternary glacial stages has been found in NW Iberia. The different magnitude of these episodes probably conditioned the distribution of currently montane species. We examined if the population history of NW Iberian Erebia triaria butterflies reflected such an influence of different glacial stages. We also investigated whether these populations were post-glacially colonised from a single refugial area or several sources. For this, we performed phylogenetic analyses and coalescence simulations on mitochondrial DNA sequences of individuals from five NW Iberian locations. We analysed three additional populations as reference, i.e., Central Spain, the Pyrenees, and the Alps. One of the NW Iberian populations, a subspecies endemic to a particular mountain range (Xistral), showed a high level of genetic divergence from all other populations, regardless of their geographic distance. Isolation after an ancient glacial stage, and followed by allopatric differentiation, may account for such high differentiation. The genetic pattern shown by the rest of the NW Iberian population samples consisted of two lineages, likely reflecting that the ancestors of these populations sought for refuge in at least two different areas during a subsequent glacial stage. We showed evidence of both temporal and spatial divisions in the phylogeography of these butterflies. The congruence of this pattern with the geological history suggests that the Iberian Peninsula hosted several refugial areas that differed in area and location depending on the glacial stage.

Animals↗

Eicosanoids mediate nodulation reactions to bacterial infections in larvae of the butterfly, Colias eurytheme.

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. Treating larvae of the butterfly Colias eurytheme with the eicosanoid biosynthesis inhibitor dexamethasone, strongly impaired nodulation reactions to bacterial infections. The influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. An eicosanoid biosynthesis system in C. eurytheme larvae is documented. Specifically, the presence of eicosanoid-precursor polyunsaturated fatty acids in tissue phospholipids was determined, an intracellular phospholipase A2 that can release arachidonic acid from tissue phospholipids was recorded, and eicosanoid biosynthesis, registered as conversion of exogenous radioactive 20:4n-6 into eicosanoids, was observed. These findings support the hypothesis that eicosanoids mediate cellular immune responses to bacterial infections in these butterfly larvae, and more broadly, in most, if not all, insects.

Animals↗

Vanessa cardui adipokinetic hormone (Vanca-AKH) in butterflies and a moth.

Small neuropeptides of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family regulate energy metabolism in insects. Within lepidopterans, the nonapeptide Manduca sexta AKH (Manse-AKH) represents a widely occurring AKH, whereas the decapeptide Helze-HrTH (at first isolated from Heliothis zea) seems to be restricted to moths. Here we show that Vanca-AKH, a non-amidated undecapeptide which we recently found in the painted lady butterfly, Vanessa cardui, is also present in the retrocerebral complex of several other butterflies (Danaus plexippus, Precis coenia, Aglais urticae) and a moth (Spodoptera frugiperda). This study also demonstrates the power of modern nano-electrospray-quadrupole TOF tandem mass spectrometry in the sequence confirmation of peptides from minute amounts of small neuropeptides.

Animals↗

Phenanthroindolizidine alkaloids as cytotoxic substances in a Danaid butterfly, Ideopsis similis, against human cancer cells.

We previously reported the presence of cytotoxic substances in extracts of the Danaid butterfly, Ideopsis similis. In the present study, we isolated cytotoxic substances against a human gastric cancer cell line, TMK-1, in I. similis pupae, with an activity similar to that of the adult butterfly. The basic fraction, prepared from a methanol extract, accounted for 83% of the cytotoxic activity. Two major cytotoxic substances were purified by HPLC, and one was determined to be a new phenanthroindolizidine alkaloid, trans-(+)-3,14alpha-dihydroxy-6,7-dimethoxyphenanthroindolizidine (1), and the other a known compound, trans-(+)-3,14alpha-dihydroxy-4,6,7-trimethoxyphenanthroindolizidine (2). The IC(50) values for TMK-1 cells were 0.5 ng/mL and 0.7 ng/mL, respectively. These two compounds showed similar cytotoxic potential with four other cancer cell lines including cervical, lung, and colon carcinomas and leukemia. Quantitative analyses indicated the presence of each of the two phenanthroindolizidine alkaloids at levels of 11-74 microg in each larva, pupa, or adult of I. similis. However, 1 was not detected in the leaves of Tylophora tanakae, a host plant for larvae of I. similis, and the level of 2 (2 microg per gram of leaves) was far less than that in the larvae. Since the leaves of T. tanakae are known to contain various phenanthroindolizidines, compounds 1 and 2 are presumably metabolically converted from such alkaloids in larvae of I. similis.

Alkaloids↗

Controlled replication of butterfly wings for achieving tunable photonic properties.

The fine structure of the wing scale of a Morpho Peleides butterfly was examined carefully, and the entire configuration was completely replicated by a uniform Al(2)O(3) coating through a low-temperature ALD process. An inverted structure was achieved by removing the butterfly wing template at high temperature, forming a polycrystalline Al(2)O(3) shell structure with precisely controlled thickness. Other than the copy of the morphology of the structure, the optical property, such as the existence of PBG, was also inherited by the alumina replica. Reflection peaks at the violet/blue range were detected on both original wings and their replica, while a simple alumina coating shifted the reflection peak to longer wavelength because of the change of periodicity and refraction index. The alumina replicas also exhibited similar functional structures as waveguide and beam splitter, which may be used as the building blocks for photonic ICs with high reproducibility and lower fabrication cost compared to traditional lithography techniques.

Aluminum Oxide↗

Plant analysis by butterflies: occurrence of cyclopentenylglycines in Passifloraceae, Flacourtiaceae, and Turneraceae and discovery of the novel nonproteinogenic amino acid 2-(3'-cyclopentenyl)glycine in Rinorea.

Following records about feeding habits of nymphalid butterflies, a novel nonproteinogenic L-amino acid, (S)-2-(3'-cyclopentenyl)glycine (11), was discovered in Rinorea ilicifolia, a species where the presence of a cyclopentanoid natural product of this kind was neither known nor anticipated from the taxonomic point of view. Another novel amino acid, (2S,1'S,2'S)-2-(2'-hydroxy-3'-cyclopentenyl)glycine (12), the stereochemistry of which was determined by single-crystal X-ray diffraction, was shown to occur in species belonging to Flacourtiaceae, Passifloraceae, and Turneraceae. These species, many of which serve as hosts for nymphalid butterflies (Acraeinae, Heliconiinae, Argynninae), also produce 2-(2'-cyclopentenyl)glycine. Cyclopentenylglycines are proposed to be novel chemical recognition templates for plant-insect interactions. Ratios between the epimers of (2S)-2-(2'-cyclopentenyl)glycine, which co-occur in plants, were determined by (1)H NMR spectroscopy. Contrary to a previous report, the (2S,1'R) epimer always appears to predominate over the (2S,1'S) epimer. Stereochemical aspects of biosynthesis of natural cyclopentanoid cyanogenic glycosides are discussed in relation to these findings.

Animals↗

Chemical defense in the zebra swallowtail butterfly, Eurytides marcellus, involving annonaceous acetogenins.

Few herbivores feed on the foliage of the North American paw paw tree, Asimina triloba; notable exceptions are the larvae of the zebra swallowtail butterfly, Eurytides marcellus. Toxic annonaceous acetogenins, produced by A. triloba, are responsible for the relative unpalatability of the leaves. Acetogenins found in A. triloba extracts are potent pesticidal and antineoplastic agents and have emetic activity in vertebrates. In this study, partitioned aqueous MeOH fractions of the bioactive CH2Cl2 extracts, of freeze-dried and pulverized larvae, and of mature butterflies revealed acetogenin content through the use of HPLC coupled to tandem MS (LC-MS/MS). This sensitive technique provides an uncomplicated method for the detection of trace compounds and, in this instance, has confirmed tissue presence of acetogenins that serve a probable role as chemical defense agents against bird predation in zebra swallowtail larvae and adults.

Animals↗

Butterfly eyespot patterns: evidence for specification by a morphogen diffusion gradient.

In this paper we describe a test for Nijhout's (1978, 1980a) hypothesis that the eyespot patterns on butterfly wings are the result of a threshold reaction of the epidermal cells to a concentration gradient of a diffusing degradable morphogen produced by focal cells at the centre of the future eyespot. The wings of the nymphalid butterfly, Bicyclus anynana, have a series of eyespots. each composed of a white pupil, a black disc and a gold outer ring. In earlier extirpation and transplantation experiments (Nijhout 1980a; French and Brakefield, 1995) it has been established that these eyespots are indeed organised around groups of signalling cells active during the first hours of pupal development. If these cells were to supply the positional information for eyespot formation in accordance with Nijhout's diffusion-degradation gradient model, then, when two foci are close together. the signals should sum, and this effect should be apparent in the detailed shape of the resulting pigment pattern. We give an equation for the form of the contours that would be obtained in this manner. We use this to test the morphogen gradient hypothesis on measurements of the outlines of fused eyespots obtained either by grafting focal cells close together, or by using a mutation (Spotty) that produces adjacent fused eyespots. The contours of the fused patterns were found to satisfy our equation, thus corroborating Nijhout's hypothesis to the extent possible with this particular type of experiment.

Animals↗

Contribution of Distal-less to quantitative variation in butterfly eyespots.

The colour patterns decorating butterfly wings provide ideal material to study the reciprocal interactions between evolution and development. They are visually compelling products of selection, often with a clear adaptive value, and are amenable to a detailed developmental characterization. Research on wing-pattern evolution and development has focused on the eyespots of the tropical butterfly Bicyclus anynana. There is quantitative variation for several features of eyespot morphology but the actual genes contributing to such variation are unknown. On the other hand, studies of gene expression patterns in wing primordia have implicated different developmental pathways in eyespot formation. To link these two sets of information we need to identify which genes within the implicated pathways contribute to the quantitative variation accessible to natural selection. Here we begin to bridge this gap by demonstrating linkage between DNA polymorphisms in the candidate gene Distal-less (Dll) and eyespot size in B. anynana.

Animals↗

The genetics and evo-devo of butterfly wing patterns.

Understanding how the spectacular diversity of colour patterns on butterfly wings is shaped by natural selection, and how particular pattern elements are generated, has been the focus of both evolutionary and developmental biologists. The growing field of evolutionary developmental biology has now begun to provide a link between genetic variation and the phenotypes that are produced by developmental processes and that are sorted by natural selection. Butterfly wing patterns are set to become one of the few examples of morphological diversity to be studied successfully at many levels of biological organization, and thus to yield a more complete picture of adaptive morphological evolution.

Animals↗

Lack of genetic differentiation among widely spaced subpopulations of a butterfly with home range behaviour.

We examine seven geographically separate subpopulations of Heliconius charithonia, a butterfly with well-documented home range behaviour, in Miami-Dade County, Florida, for genetic differentiation using cellulose acetate electrophoresis. These subpopulations exhibit little genetic variation (percent polymorphic loci = 27, average heterozygosity = 0.103) especially in comparison to populations of the same and related species from mainland South America. Allele frequencies do not differ among the subpopulations in south Florida and estimates of Wright's fixation index (FST) support that there is no detectable genetic differentiation among them. This result supports an earlier finding that the dispersal ability of Heliconius butterflies may be underestimated. However, it is unlikely that increased dispersal ability alone could account for the lack of genetic differentiation observed among subpopulations separated by almost 80 km. Given the likely effective population size of these subpopulations (Ne = 205) and the average generation time of this species in the subtropics (in the range of 30-90 days), this lack of genetic differentiation is best explained by current or very recent gene flow following a stepping-stone model. Furthermore, this result provides evidence that the current extensive degree of habitat fragmentation surrounding the city of Miami does not limit gene flow among urban subpopulations of Heliconius charithonia.

Alleles↗

Lack of response to artificial selection on the slope of reaction norms for seasonal polyphenism in the butterfly Bicyclus anynana.

The tropical butterfly Bicyclus anynana shows adaptive phenotypic plasticity in response to wet-dry seasonality. The wet season form (WSF) has a conspicuous wing pattern with large eyespots, whereas the dry season form (DSF) lacks eyespots and therefore has a more cryptic appearance. Temperature is the main factor controlling this difference: rearing larvae at a low (<19 degrees C) temperature in the laboratory results in the DSF, whereas rearing at a high (>23 degrees C) temperature induces the WSF. We applied truncation selection in opposite directions in successive generations reared at two alternating temperatures (18.5 degrees C and 23.5 degrees C) to increase (for two High Plasticity (HP) lines), and decrease (for two Low Plasticity (LP) lines) wing pattern plasticity. Plasticity was assessed by partitioning full-sib families over four rearing temperatures (18.5 degrees C, 20.5 degrees C, 21.5 degrees C and 23.5 degrees C). Several wing pattern elements were measured for which the first principal component (PC1) provides a useful summary. The slopes of reaction norms for PC1 were significantly steeper in the HP lines than in the LP lines; however, the selection lines did not always differ significantly from the unselected stock. The results of crosses between the replicates of the selection lines gave no indication for effects of inbreeding. We argue that high, positive genetic correlations across temperatures retard a response to selection in opposite directions in different environments. This is discussed with respect to potential evolutionary constraints in natural populations in these butterflies.

Adaptation, Biological↗

Patterns of mitochondrial versus nuclear DNA sequence divergence among nymphalid butterflies: the utility of wingless as a source of characters for phylogenetic inference.

To investigate the utility of a region of wingless (wg) as a marker for molecular systematics, we compared wg sequences to mitochondrial COII sequences from twenty-two nymphalid butterfly taxa and one outgroup. Compositional characteristics of the two gene regions are compared, and their contributions to a cladogram inferred from the combined data set are assessed. Primarily due to its uniform base composition, wg appears to become saturated more slowly than mtDNA, although the two genes appear to be evolving at quite similar rates. We suggest that wg will be a useful source of characters for phylogenetic studies of butterflies, and perhaps other insect taxa, with divergence times up to 60 million years ago.

Animals↗

Microsatellite markers to assess the influence of population size, isolation and demographic change on the genetic structure of the UK butterfly Polyommatus bellargus.

Five microsatellite DNA markers were isolated and used to quantify population genetic structure among a subset of UK populations of the Adonis blue (Polyommatus bellargus Rottemburg). Specifically, whether population size, degree of isolation or history of bottlenecking in 1976-1978 can explain current patterns of genetic variation. The butterfly is at its northern range limit in the UK, where it exists as a highly fragmented metapopulation on isolated pockets of calcareous grassland. Most populations were affected by a severe bottleneck in the late 1970s, when a drought caused the host plant (Hippocrepis comosa) to wilt. Mantel tests and spatial autocorrelation analysis indicated a significant effect of isolation by distance among the UK populations, a relationship that broke down at greater geographical scales (> 23.85 km), probably because of large areas of unsuitable habitat presenting barriers to gene flow. Similarly, amova revealed that variation among geographical regions was almost double that observed within regions. Larger populations were found to support significantly higher levels of genetic diversity, suggesting that small populations may lose genetic diversity through drift. If, as in other butterfly species, low genetic diversity increases the probability of population extinction, then these populations are likely to be under threat. Neither isolation nor a history of bottlenecks appeared to influence genetic diversity. The results indicate that adequate population size a crucial factor in the conservation of genetic diversity in P. bellargus in the UK.

Alleles↗