Baiting Insects with Pyrrolizidine Alkaloids (PAs): A Fieldwork-Oriented Review and Guide to PA-Pharmacophagy.

Since 1890, many observations of danaine butterflies visiting dry plants of several families in the Old and New World tropics have been published. For 50 years, it has been recognised that Danainae, along with various other insects, seek out 1,2-dehydropyrrolizidine ester alkaloids (PAs) independently of and in addition to their nutritive requirements and utilise them to increase their chances for survival and biological fitness. This represents an unusual type of insect-plant relationship ("PA-pharmacophagy"), with remarkable peculiarities but also with gaps in knowledge, many of which can be filled by employing PA-baiting. We review and analyse the history of records on the attraction of adult insects to PAs and unveil the complex background information on PA-chemistry, PA-producing plants ("PA-plants"), and PA-sequestering insects ("PA-insects") in order to come up with practical tips for successful baiting with PAs ("PA-baiting"). Studying PA-pharmacophagy integrates taxonomy, behaviour, and ecology with evolutionary biology, chemistry, and toxicology. With basic knowledge of PA-chemistry and bearing the general peculiarities of PA-plants and PA-insects in mind, PA-baiting can be conducted easily and successfully to address many questions on the multifaceted ecology of pyrrolizidine alkaloids. We aim to encourage field researchers in the tropics to employ PA-baiting as a valuable research method in this field of integrative biology.

Immature Stages, Natural History, Systematics and Conservation of an Endangered Neotropical Butterfly: the Case of Scada karschina delicata (Nymphalidae: Ithomiini).

The endangered butterfly Scada karschina delicata Talbot, 1932 (Nymphalidae: Danainae: Ithomiini) is endemic to northeastern Brazil, occurring in very few forest remnants of the 'Pernambuco Center of Endemism'. Larvae feed on Solanaceae and are very similar to those of other species in the subtribe Mechanitina, with lateral projections on body, one of the main synapomorphies of this subtribe. Based on molecular data, S. k. delicata clustered together with S. karschina karschina, as a monophyletic group sister to the amazon clade of S. reckia. Based on all available data, S. k. delicata is known from only five localities of mid- to high-altitude forests (from 500 to 1000 m of altitude) in northeastern Brazil. Grounded on available data, a new assessment of extinction risk is proposed, and S. k. delicata is now considered Vulnerable (VU) taxon.

Linking karyotypes with DNA barcodes: proposal for a new standard in chromosomal analysis with an example based on the study of Neotropical Nymphalidae (Lepidoptera).

Chromosomal data are important for taxonomists, cytogeneticists and evolutionary biologists; however, the value of these data decreases sharply if they are obtained for individuals with inaccurate species identification or unclear species identity. To avoid this problem, here we suggest linking each karyotyped sample with its DNA barcode, photograph and precise geographic data, providing an opportunity for unambiguous identification of described taxa and for delimitation of undescribed species. Using this approach, we present new data on chromosome number diversity in neotropical butterflies of the subfamily Biblidinae (genus Vila Kirby, 1871) and the tribe Ithomiini (genera Oleria Hübner, 1816, Ithomia Hübner, 1816, Godyris Boisduval, 1870, Hypothyris Hübner, 1821, Napeogenes Bates, 1862, Pseudoscada Godman et Salvin, 1879 and Hyposcada Godman et Salvin, 1879). Combining new and previously published data we show that the species complex Oleria onega (Hewitson, [1852]) includes three discrete chromosomal clusters (with haploid chromosome numbers n = 15, n = 22 and n = 30) and at least four DNA barcode clusters. Then we discuss how the incomplete connection between these chromosomal and molecular data (karyotypes and DNA barcodes were obtained for different sets of individuals) complicates the taxonomic interpretation of the discovered clusters.

Two types of highly ordered micro- and macrochromosome arrangement in metaphase plates of butterflies (Lepidoptera).

In karyotype of many organisms, chromosomes form two distinct size groups: macrochromosomes and microchromosomes. During cell divisions, the position of the macro- and microchromosomes is often ordered within metaphase plate. In many reptiles, amphibians, birds, insects of the orthopteran family Tettigoniidae and in some plants, a so called "reptilian" type organization is found, with microchromosomes situated in the center of metaphase plate and with macrochromosomes situated at the periphery. An opposite, "lepidopteran" type is known in butterflies and moths (i.e. in the order Lepidoptera) and is characterized by macrochromosomes situated in the center and by microchromosomes situated at the periphery. The anomalous arrangement found in Lepidoptera was previously explained by holocentric organization of their chromosomes. Here I analyse the structure of meiotic metaphase I plates in ithomiine butterfly, Forbestraolivencia (H. Bates, 1862) (Nymphalidae, Danainae, Ithomiini) which has a clear "reptilian" organization, contrary to previous observations in Lepidoptera. In this species large bivalents (i.e. macrochromosomes) form a regular peripheral circle, whereas the minute bivalents (i.e. microchromosomes) occupy the center of this circle. The reasons and possible mechanisms resulting in two drastically different spatial chromosome organization in butterflies are discussed.

HPLC-MS detection of pyrrolizidine alkaloids and their N-oxides in herbarium specimens dating back to the 1850s.

PREMISE OF THE STUDY: Understanding the phylogenetic distribution of defensive plant secondary metabolites is essential to the macroevolutionary study of chemically mediated plant-animal interactions. The chemical ecology of pyrrolizidine alkaloids (PAs) has been extensively studied in a number of plant-herbivore systems, including Apocynaceae (the milkweed and dogbane family) and Danainae (the milkweed and clearwing butterflies). A systematic survey is necessary to establish a detailed understanding of their occurrence across Apocynaceae. A survey of this species-rich, mainly tropical and subtropical family will rely heavily on small tissue samples removed from herbarium specimens, some of which will be very old and/or preserved with alcohols or mercuric chloride. METHODS: We optimized PA extraction methods from small leaf fragments of recently collected silica-dried leaves of the PA-positive Echites umbellatus, varying crushing and extraction time. We then applied our optimized method to leaf fragments from 70-167-year-old herbarium specimens of E. umbellatus. To simulate the effect of alcohol treatment on PA detectability in herbarium specimens, we incubated freshly collected leaves of the PA-positive Parsonsia alboflavescens in three different alcohols before drying and compared PA recovery to freshly dried controls. PAs were quantified using high-performance liquid chromatography-mass spectrometry analysis. X-ray fluorescence was used to identify mercury-containing specimens. RESULTS: Fifteen seconds of leaf crushing followed by 24 h of extraction were optimal for PA free-base and N-oxide recovery. This method yielded ~50-fold greater PA recovery than prior methods. Half of the herbarium specimens (13 of 23), including the oldest, tested positive for PAs; leaf age did not correlate with success in PA extraction. Treatment of fresh leaves with alcohol before drying did not diminish PA recovery; mercury was observed in both PA-positive and PA-negative specimens. CONCLUSIONS: PAs can be reliably detected in small tissue samples from herbarium specimens up to 167 years old, including specimens that had been treated with alcohol or mercury salts. The variability of PA presence among herbarium specimens of E. umbellatus indicates that multiple specimens will need to be tested before a particular species is determined to lack PAs.

Does spatial variation in predation pressure modulate selection for aposematism?

It is widely believed that aposematic signals should be conspicuous, but in nature, they vary from highly conspicuous to near cryptic. Current theory, including the honest signal or trade-off hypotheses of the toxicity-conspicuousness relationship, cannot explain why adequately toxic species vary substantially in their conspicuousness. Through a study of similarly toxic Danainae (Nymphalidae) butterflies and their mimics that vary remarkably in their conspicuousness, we show that the benefits of conspicuousness vary along a gradient of predation pressure. Highly conspicuous butterflies experienced lower avian attack rates when background predation pressure was low, but attack rates increased rapidly as background predation pressure increased. Conversely, the least conspicuous butterflies experienced higher attack rates at low predation pressures, but at high predation pressures, they appeared to benefit from crypsis. Attack rates of intermediately conspicuous butterflies remained moderate and constant along the predation pressure gradient. Mimics had a similar pattern but higher attack rates than their models and mimics tended to imitate the signal of less attacked model species along the predation pressure gradient. Predation pressure modulated signal fitness provides a possible mechanism for the maintenance of variation in conspicuousness of aposematic signals, as well as the initial survival of conspicuous signals in cryptic populations in the process of aposematic signal evolution, and an alternative explanation for the evolutionary gain and loss of mimicry.

The complete mitochondrial genome of Tirumala limniace (Lepidoptera: Nymphalidae: Danainae).

The complete mitochondrial nucleotide sequence of Tirumala limniace is 15,285 bp in size, containing typical metazoan 37 genes: 13 protein-coding genes (PCGs), 22tRNA genes, 2 ribosomal RNA genes and one noncoding AT-rich region. All the 13 PCGs initiated with ATN, except for COI gene which is started by CGA codon. Nine PCGs use the complete termination codon (TAA), whereas the COI, COII, ND4 and ND5 genes end with single T. A total of 132 bp intergenic spacers and a total of 37 bp overlapping sequences are interspersed throughout the whole genome. The two rRNA genes (rrnL and rrnS) are 1352 bp and 779 bp in size, with their AT contents of 84.6% and 85.5%, respectively. All tRNA genes display typical secondary cloverleaf structures except for trnS1 (AGN) which loses DHU arm. The 438 bp long AT-rich region contains several features common to the other lepidopterans, such as the ATAGA motif followed by a 18 bp poly-T stretch, a 9 bp poly-T stretch, a 10 bp poly-A stretch and some microsatellite-like (AT)n elements.

The complete mitochondrial genome of Ideopsis similis (Lepidoptera: Nymphalidae: Danainae).

The complete mitochondrial nucleotide sequence of Ideopsis similis is 15,200 bp in size, containing typical metazoan 37 genes: 13 protein-coding genes (PCGs), 22 tRNA genes, 2 ribosomal RNA genes and 1 non-coding AT-rich region. All the 13 PCGs initiated with ATN, except for COI gene which started by CGA codon. Nine PCGs used the complete termination codon (TAA), whereas the COI, COII, ND4 and ND5 genes ended with single T. The two rRNA genes (rrnL and rrnS) were 1318 bp and 786 bp in size, with their AT contents of 84.7% and 85.7%, respectively. All tRNA genes display typical secondary cloverleaf structures except for trnS1(AGN) which loses DHU arm. The 404 bp long AT-rich region contains several features common to the other lepidopterans, such as the ATAGA motif followed by a 19 bp poly-T stretch, a 9 bp poly-T stretch and some microsatellite-like (AT)n elements.

Incompatible Ages for Clearwing Butterflies Based on Alternative Secondary Calibrations.

The recent publication of a time-tree for the plant family Solanaceae (nightshades) provides the opportunity to use independent calibrations to test divergence times previously inferred for the diverse Neotropical butterfly tribe Ithomiini. Ithomiini includes clades that are obligate herbivores of Solanaceae, with some genera feeding on only one genus. We used 8 calibrations extracted from the plant tree in a new relaxed molecular-clock analysis to produce an alternative temporal framework for the diversification of ithomiines. We compared the resulting age estimates to: (i) a time-tree obtained using 7 secondary calibrations from the Nymphalidae tree of Wahlberg et al. (2009), and (ii) Wahlberg et al.'s (2009) original age estimates for the same clades. We found that Bayesian clock estimates were rather sensitive to a variety of analytical parameters, including taxon sampling. Regardless of this sensitivity however, ithomiine divergence times calibrated with the ages of nightshades were always on average half the age of previous estimates. Younger dates for ithomiine clades appear to fit better with factors long suggested to have promoted diversification of the group such as the uplifting of the Andes, in the case of montane genera. Alternatively, if ithomiines are as old as previous estimates suggest, the recent ages inferred for the diversification of Solanaceae seem likely to be seriously underestimated. Our study exemplifies the difficulty of testing hypotheses of divergence times and of choosing between alternative dating scenarios, and shows that age estimates based on seemingly plausible calibrations may be grossly incongruent.

The complete mitochondrial genome of Danaus chrysippus (Lepidoptera: Nymphalidae: Danainae).

The complete mitochondrial genome sequence of Danaus chrysippus (Lepidoptera: Nymphalidae: Danainae) was determined. The 15,236 bp long genome encodes 13 putative proteins, two ribosomal RNAs, 22 tRNAs and a non-coding AT-rich region. Its gene arrangement pattern is identical to most of other lepidopteran species. All protein-coding genes start with a typical ATN codon with the exception of COI gene which uses CGA as its initial codon; all PCGs terminate in the common stop TAA or TAG, except COI, COII, ND5 and ND4 which use single T as their stop codons. A total of 102 bp intergenic spacers and a total of 33 bp overlapping sequences are interspersed throughout the whole genome. The mitogenome harbors 22 txRNAs as those of most insect species and all tRNA genes evidence the typical clover leaf secondary structures with the exception of tRNAser (AGN) who loses its dihydrouridine (DHU) arm. The lrRNA and srRNA genes are 1339 and 783 bp, with the AT contents of 84.1 and 84.8%, respectively. The non-coding AT-rich region is 418 bp long, and contains the motif ATAGA followed by a 21-bp poly-T stretch and a microsatellite-like (AT)9 element preceded by the ATTTA motif.

Stepwise evolution of resistance to toxic cardenolides via genetic substitutions in the Na+/K+ -ATPase of milkweed butterflies (lepidoptera: Danaini).

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na(+)/K(+) -ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so-called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide-containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na(+)/K(+) -ATPase gene and by enzymatic assays with extracted Na(+)/K(+) -ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom-up selection by plant compounds and top-down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.