Swallowtail Butterflies Use Multiple Visual Cues to Select Oviposition Sites

Flower-foraging Japanese yellow swallowtail butterflies, Papilio xuthus, exhibit sophisticated visual abilities. When ovipositing, females presumably attempt to select suitable leaves to support the growth of their larval offspring. We first established that butterflies indeed select particular leaves on which to lay eggs; when presented with a single Citrus tree, butterflies significantly favored two out of 102 leaves for oviposition. These preferences were observed across many individuals, implying that they were not merely idiosyncratic, but rather based on properties of the leaves in question. Because the butterflies descended towards the leaves rather directly from a distance, we hypothesized that they base their selection on visual cues. We measured five morphological properties (height, orientation, flatness, roundness, and size) and four reflective features (green reflectance, brightness, and degree and angle of linear polarization). We found that the number of eggs laid upon a leaf was positively correlated with its height, flatness, green reflectance, and brightness, and negatively correlated with its degree of polarization, indicating that these features may serve as cues for leaf selection. Considering that other studies report ovipositing butterflies’ preference for green color and horizontally polarized light, butterflies likely use multiple visual features to select egg-laying sites on the host plant.

Genome-wide identification and gene-editing of pigment transporter genes in the swallowtail butterfly Papilio xuthus

Background Insect body coloration often functions as camouflage to survive from predators or mate selection. Transportation of pigment precursors or related metabolites from cytoplasm to subcellular pigment granules is one of the key steps in insect pigmentation and usually executed via such transporter proteins as the ATP-binding cassette (ABC) transmembrane transporters and small G-proteins (e.g. Rab protein). However, little is known about the copy numbers of pigment transporter genes in the butterfly genomes and about the roles of pigment transporters in the development of swallowtail butterflies. Results Here, we have identified 56 ABC transporters and 58 Rab members in the genome of swallowtail butterfly Papilio xuthus. This is the first case of genome-wide gene copy number identification of ABC transporters in swallowtail butterflies and Rab family in lepidopteran insects. Aiming to investigate the contribution of the five genes which are orthologous to well-studied pigment transporters (ABCG: white, scarlet, brown and ok; Rab: lightoid) of fruit fly or silkworm during the development of swallowtail butterflies, we performed CRISPR/Cas9 gene-editing of these genes using P. xuthus as a model and sequenced the transcriptomes of their morphological mutants. Our results indicate that the disruption of each gene produced mutated phenotypes in the colors of larvae (cuticle, testis) and/or adult eyes in G0 individuals but have no effect on wing color. The transcriptomic data demonstrated that mutations induced by CRISPR/Cas9 can lead to the accumulation of abnormal transcripts and the decrease or dosage compensation of normal transcripts at gene expression level. Comparative transcriptomes revealed 606 ~ 772 differentially expressed genes (DEGs) in the mutants of four ABCG transporters and 1443 DEGs in the mutants of lightoid. GO and KEGG enrichment analysis showed that DEGs in ABCG transporter mutants enriched to the oxidoreductase activity, heme binding, iron ion binding process possibly related to the color display, and DEGs in lightoid mutants are enriched in glycoprotein binding and protein kinases. Conclusions Our data indicated these transporter proteins play an important role in body color of P. xuthus. Our study provides new insights into the function of ABC transporters and small G-proteins in the morphological development of butterflies.

Genome-wide macroevolutionary signatures of key innovations in butterflies colonizing new host plants

The mega-diversity of herbivorous insects is attributed to their co-evolutionary associations with plants. Despite abundant studies on insect-plant interactions, we do not know whether host-plant shifts have impacted both genomic adaptation and species diversification over geological times. We show that the antagonistic insect-plant interaction between swallowtail butterflies and the highly toxic birthworts began 55 million years ago in Beringia, followed by several major ancient host-plant shifts. This evolutionary framework provides a valuable opportunity for repeated tests of genomic signatures of macroevolutionary changes and estimation of diversification rates across their phylogeny. We find that host-plant shifts in butterflies are associated with both genome-wide adaptive molecular evolution (more genes under positive selection) and repeated bursts of speciation rates, contributing to an increase in global diversification through time. Our study links ecological changes, genome-wide adaptations and macroevolutionary consequences, lending support to the importance of ecological interactions as evolutionary drivers over long time periods.

Comparison of flower-visiting behaviour of bumblebees and swallowtail butterflies to the Japanese azalea (Rhododendron japonicum)

Examination of the efficacy of pollinators that are not morphologically matched to a flower’s shape can deepen our understanding of the main pollinators of a plant species. In central Japan, Rhododendron japonicum is visited much more frequently by bumblebees than by butterflies although its flower shape is more suited for pollination by large butterflies. Here, we observed flower-visiting behaviour of Bombus diversus queens and the alpine black swallowtail butterfly, Papilio maackii, and compared their efficacy as pollinators. Papilio maackii always touched the stigma during a flower visit, whereas B. diversus queens did so during just 54% of their flower visits. As bumblebees visit neighboring flowers of a R. japonicum tree in sequence not like Papilio maackii, we hypothesized that they deliver self pollen to R. japonicum so that the fruit set would be low. However, the fruit set in a bumblebee-dominated area was 72.5%, significantly higher than that by hand-pollination with self pollen (31.4%). This suggests that R. japonicum can obtain sufficient fruits by B. diversus queens although its flower shape morphologically matches to pollination by swallowtail butterflies.

Genome-wide macroevolutionary signatures of key innovations in butterflies colonizing new host plants

The exuberant proliferation of herbivorous insects is attributed to their associations with plants. Despite abundant studies on insect-plant interactions, we do not know whether host-plant shifts have impacted both genomic adaptation and species diversification over geological times. We show that the antagonistic insect-plant interaction between swallowtail butterflies and the highly toxic birthworts began 55 million years ago in Beringia, followed by several major ancient host-plant shifts. This evolutionary framework provides a unique opportunity for repeated tests of genomic signatures of macroevolutionary changes and estimation of diversification rates across their phylogeny. We find that host-plant shifts in butterflies are associated with both genome-wide adaptive molecular evolution (more genes under positive selection) and repeated bursts of speciation rates, contributing to an increase in global diversification through time. Our study links ecological changes, genome-wide adaptations and macroevolutionary consequences, lending support to the importance of ecological interactions as evolutionary drivers over long time periods.

Swallowtail butterflies (Lepidoptera: Papilionidae) species diversity and distribution in Africa: The Papilionidae collection at the National Museums of Kenya, Nairobi, Kenya

Species data from the Museum collections have been shown to be of great value as a tool for prioritising conservation actions in Africa (Fjeldsa and Tushabe 2005). The National Museums of Kenya (NMK) have an entomology collection, housed in 4,000 drawers in cabinets that contain over 1.5 million specimens, including the largest butterfly collection in Africa (Arnett et al. 1997). Lampe and Striebing (2005) demonstrated how to digitise large insect collections in order to make their associated label data into databases that can be used for functions, such as creating distribution maps. The NMK’s swallowtail butterflies' collection had not been digitised and thus there was a need to capture the label data to create a database that can be used for mapping the distribution of the species in Kenya and elsewhere. These data have addressed one of the most significant challenges to insect conservation i.e. the lack of baseline information concerning species diversity and distribution (Summerville and Crist 2003). These data have provided key historic papilionid species diversity and distribution data that can be used to monitor their populations, as butterflies are declining due to changes in land use, intensive agriculture and pestcide use, diseases and pest and climate change (Potts et al. 2016; Bongaarts 2019). The publication of the occurrence data records in GBIF has been undertaken, thus making the data available to a wider audience and promoting availability for use. The swallowtail butterflies collection at the National Museums of Kenya was digitised from 2017–2019 and this paper presents details of the Papilionid collection at the Zoology Department, NMK, Nairobi, Kenya. The collection holds 7,345 voucher specimens, consisting of three genera and 133 species. The collection covers the period between 1850 to 2019. The distribution of the swallowtail butterflies, housed at the NMK, covers East Africa with 88%, Central Africa (6%), Western Africa (4%) and Southern Africa (2%).

Cold Stratification and Pericarp Removal Improve Seed Germination of Ptelea trifoliata and Ptelea crenulata

Two species of North American shrubs in the genus Ptelea (Rutaceae), commonly known as eastern and western hoptree or wafer ash, have unfulfilled potential to increase the diversity of managed landscapes and support populations of pollinators and swallowtail butterflies. The white flowers of Ptelea are highly fragrant, and pistillate flowers give rise to clusters of distinctive samaras. The insufficiency of information about improving germination of seeds of Ptelea trifoliata and lack of recommendations for Ptelea crenulata prompted us to investigate effects of pericarp removal and cold (4 °C) stratification periods of 0, 4, 8, and 16 weeks on the germination of seeds of these two species. Samaras were collected from multiple plants of both species in the midwestern United States (P. trifoliata) and California (P. crenulata). The germination percentage of viable seeds, calculated after tests of viability with tetrazolium chloride of seeds that did not germinate, increased with longer stratification periods, with 100% germination for P. crenulata and 91% germination for P. trifoliata after 16 weeks of stratification. The germination value, a measure of the speed and uniformity of germination, and peak value also increased with longer stratification in both species. Pericarp removal increased the germination percentage of both species and increased the peak and germination value of P. crenulata. Propagators seeking to grow these species of Ptelea from seed should remove the pericarp and cold-stratify seeds for 16 weeks to improve germination success.