Polarization-based brightness discrimination in the foraging butterfly, Papilio xuthus

The human eye is insensitive to the angular direction of the light e-vector, but several animal species have the ability to discriminate differently polarized lights. How the polarization is detected is often unclear, however. Egg-laying Papilio butterflies have been shown to see false colours when presented with differently polarized lights. Here we asked whether this also holds in foraging butterflies. After training individuals to feed on nectar in front of an unpolarized spectral light, we carried out three dual-choice tests, where the discrimination of (i) the spectral content, (ii) the light intensity, and (iii) the e-vector orientation were investigated. In the first test, the butterflies selected the trained spectrum irrespective of its intensity, and in the second test they chose the light with the higher intensity. The result of the e-vector discrimination test was very similar to that of the second test, suggesting that foraging butterflies discriminate differently polarized lights as differing in brightness rather than as differing in colour. Papilio butterflies are clearly able to use at least two modes of polarization vision depending on the behavioural context.     

The butterfly Papilio xuthus detects visual motion using chromatic contrast

Many insects’ motion vision is achromatic and thus dependent on brightness rather than on colour contrast. We investigate whether this is true of the butterfly Papilio xuthus, an animal noted for its complex retinal organization, by measuring head movements of restrained animals in response to moving two-colour patterns. Responses were never eliminated across a range of relative colour intensities, indicating that motion can be detected through chromatic contrast in the absence of luminance contrast. Furthermore, we identify an interaction between colour and contrast polarity in sensitivity to achromatic patterns, suggesting that ON and OFF contrasts are processed by two channels with different spectral sensitivities. We propose a model of the motion detection process in the retina/lamina based on these observations.     

A novel retinol-binding protein in the retina of the swallowtail butterfly, Papilio xuthus.

Retinoid-binding proteins are indispensable for visual cycles in both vertebrate and invertebrate retinas. These proteins stabilize and transport hydrophobic retinoids in the hydrophilic environment of plasma and cytoplasm, and allow regeneration of visual pigments. Here, we identified a novel retinol-binding protein in the eye of a butterfly, Papilio xuthus. The protein that we term Papilio retinol-binding protein (Papilio RBP) is a major component of retinal soluble proteins and exclusively binds 3-hydroxyretinol, and emits fluorescence peaking at 480 nm under ultraviolet (UV) illumination. The primary structure, deduced from the nucleotide sequence of the cDNA, shows no similarity to any other lipophilic ligand-binding proteins. The molecular mass and isoelectric point of the protein estimated from the amino-acid sequence are 26.4 kDa and 4.92, respectively. The absence of any signal sequence for secretion in the N-terminus suggests that the protein exists in the cytoplasmic matrix. All-trans 3-hydroxyretinol is the major ligand of the Papilio RBP in dark-adapted eyes. Light illumination of the eyes increases the 11-cis isomer of the ligand and induces redistribution of the Papilio RBP from the proximal to the distal part of the photoreceptor layer. These results suggest that the Papilio RBP is involved in visual pigment turnover.     

Action spectrum of foraging behavior of the Japanese yellow swallowtail butterfly, Papilio xuthus

This paper describes the action spectrum of foraging behavior of a butterfly, Papilio xuthus. We first established an experimental protocol to evaluate learning and discrimination of monochromatic light by the butterflies. We trained butterflies to feed on sucrose solution at the window illuminated with certain monochromatic light produced through a monochromator. After confirming that they learned the monochromatic light, after 10 days of training, we tested the butterflies one by one. We presented training wavelengths for each individual at different intensities, and recorded whether they perform foraging behavior under freely-flying as well as tethered conditions. Freely-flying butterflies responded to light by visiting the window and searching for nectar around it, whereas tethered butterflies responded by extending their proboscides towards the window. The light intensity required to elicit 50% response for each tested monochromatic light was plotted. The resulting action spectrum for the visit was rather flat with the maximum sensitivity a 420 nm, whereas the spectrum for the proboscis extension had prominent peaks at 380, 500 and 600 nm. The difference in action spectra indicates that the visit and the proboscis extension are controlled by two independent mechanisms at least in part.     

Population dynamics of the swallowtail butterfly,Papilio xuthus L., in a deforested area

The long-term population dynamics of the swallowtail butterfly, Papilio xuthus, were studied by means of life table analysis in a deforested area, where the host tree, Zanthoxylum ailanthoides, was growing. The number of eggs laid on host trees in the deforested area decreases as secondary succession progresses. When the trees were classified into three groups, i. e. tall, medium and short, according to their height relative to the surrounding vegetation, less eggs were laid on tall than on short host trees. Life tables for the natural populations in each generation were developed on the basis of mean values for 6 years. Eggs and larvae in early stages were attacked chiefly by small- and middle-sized predators, such as ants, spiders, bugs and orthopterids, while later stage larvae were attacked chiefly by birds or Polistes wasps. An inchneumonid wasp was the most important mortality factor in the pupal stage. Key-factor analysis was tested on the life table data of each immature stage. The late larval stage was a key stage, and most mortality factors were considered to operate independently. The test for density dependency did not show any tendencies. The analysis of variance in two-way classifications was carried out for the difference of survival rate among the tree groups. It was suggested that tall host trees were unsuitable for P. xuthus. The construction of life tables for artificially inoculated populations on large host trees showed that high physiological death rate were characteristic of such trees in comparison with natural populations on smaller trees. The characteristics of the population dynamics of P. xuthus in the deforested area are compared among some kinds of the habitat, and the interrelationship between P. xuthus and its host plant are discussed.     

Properties of Orange-Pupa-Inducing Factor (OPIF) in the swallowtail butterfly, Papilio xuthus L

Diapause pupae of the swallowtail butterfly Papilio xuthus L. exhibit diapause-green, orange and brownish-orange color polymorphism. Development of orange pupae involves a neuroendocrine factor inducing orange pupa (Orange-Pupa-Inducing Factor, OPIF), which is secreted from the head-thoracic region during late pharate pupal stages, in particular from the ganglia of short-day animals located posteriorly from the second thoracic ganglion2 (TG2). This report describes certain properties of OPIF using bioassays involving ligated abdomens of short-day pharate pupae. Localization of OPIF in the central nervous system of short-day larvae indicated that it was present predominantly in TG2, thoracic ganglion3 (TG3) and abdominal ganglion1 (AG1) complexes. OPIF activity in TG(2,3)-AG1 complexes was over two times higher than in the more posteriorely located ganglia. The developmental profile of OPIF in last instar short-day larvae revealed that OPIF activity in larval ganglia posterior to TG2 became gradually higher as larval growth proceeded, suggesting that OPIF might be accumulated in TG(2,3)-AG(1-7) complexes as larvae prepare for pupal molting. Furthermore, ligated abdomens of short-day larvae developed into pupae of an orange type when a 2% NaCl extract containing OPIF prepared from TG(2,3)-AG(1-7) complexes of long-day larvae was injected into ligated abdomens of short-day pharate pupae, indicating that OPIF is also present in long-day larvae. Additionally, a biochemical investigation using gel filtration chromatography showed that the molecular weight of OPIF was about 10 kDa.     

Interactions of environmental factors influencing pupal coloration in swallowtail butterfly Papilio xuthus

The swallowtail butterfly Papilio xuthus Linné [Lepidoptera: Papilionidae] exhibits pupal protective color polyphenism. Interactions of various environmental factors on pupal coloration were analyzed in non-diapausing individuals. Under sufficient light (200lux), most pupating larvae became green pupae when the surface of the pupation site was smooth, while they became brown when the surface was rough. Tactile signals are the positive environmental factors causing induction of the brown pupal coloration. In dark boxes, the induction of the brown pupal coloration was easily induced even on a smooth surface, suggesting that light suppresses induction of brown coloration. Different colors of pupation sites did not affect pupal coloration under sufficient light. Environmental factors received during a critical period both before girdling and after girdling affected pupal coloration. When tactile signals received from rough surfaces reach threshold levels during pupation, brown pupal coloration is determined. Larvae reared under a daily periodicity of natural light formed a girdle at midnight, subsequently, the prepupae received strong daylight the following day. Under natural light most larvae produced brown pupae on rough surfaces and green pupae on smooth surfaces.     

Multiple matings increase the fecundity of the yellow swallowtail butterfly,Papilio xuthus L., in summer generations

Females of the yellow swallowtail butterfly, Papilio xuthus,were reared in the laboratory. They were divided into four groups held under different mating conditions: nonmating (virgin) and mated once, twice, and three times. The number of eggs in the ovaries was counted by dissection. Virgin females produced increasing numbers of mature eggs, up to about 30, in the week following emergence. When the female had mated once, the number of mature eggs was significantly higher than that of virgin females by the second day after emergence. However, the double- and triplemated females did not increase the number of eggs in each state further than the singlemated females. The double-mated females deposited significantly more eggs than the singlemated females in the laboratory. The triplemated females also deposited more eggs on the day after the third mating than the doublemated females. Thus, multiple matings increased the number of eggs deposited. The change in the hatchability and the morphology of the spermatophore in the bursa copulatrix suggested that the sperm from the last mating had precedence.     

Perturbation of the wing color pattern of a swallowtail butterfly,Papilio xuthus,induced by acid carboxypeptidase

Hypodermal injection of Toughmac-E, a digestive mixture composed of nine digestive components, or Molsin induced perturbation of the wing color pattern in 0- to 2-day-old pupae of Papilio xuthus, but had no effect on prepupae or 3- to 4-day-old pupae. The effective component in Toughmac-E was identified as Molsin, an acid carboxypeptidase of Aspergillus saitoi which specifically liberates tyrosine and phenylalanine from the C-terminal residues of proteins. The pattern perturbation occurred in either side of the fore- and hindwings of both sexes. When this enzyme was administered, stronger melanization than in the normal wings was found in the whole wings of most butterflies, but in other butterflies, the yellow region was enlarged. These findings suggest that the pattern perturbation was caused by changes in the levels of melanin and papiliochromes in scales. Melanin is a black pigment and papiliochromes are yellow pigments; their common precursor is dopamine. The normal pattern is formed by a predetermined balance of melanin and papiliochromes, whereas the deposit of an excess amount of tyrosine and/or phenylalanine disturbs this balance and results in perturbation of the color pattern. Administration of L-dopa or dopamine had no effect on the wing pattern. When the activity of an endogenous acid carboxypeptidase similar to Molsin appears in the early pupa, the summed activities of the endogenous and exogenous acid carboxypeptidases must induce a pattern perturbation. The relations between the endogenous acid carboxypeptidase and its probable substrate, the reserve protein, and the physiological roles of these relations in the regulation, utilization and excretion of amino acids are discussed.     

Purification and characterization of biliverdin-binding protein from larval hemolymph of the swallowtail butterfly, Papilio xuthus L

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The Namino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.     

Structure and function of papiliocin with antimicrobial and anti-inflammatory activities isolated from the swallowtail butterfly, Papilio xuthus

Papiliocin is a novel 37-residue cecropin-like peptide isolated recently from the swallowtail butterfly, Papilio xuthus. With the aim of identifying a potent antimicrobial peptide, we tested papiliocin in a variety of biological and biophysical assays, demonstrating that the peptide possesses very low cytotoxicity against mammalian cells and high bacterial cell selectivity, particularly against Gram-negative bacteria as well as high anti-inflammatory activity. Using LPS-stimulated macrophage RAW264.7 cells, we found that papiliocin exerted its anti-inflammatory activities by inhibiting nitric oxide (NO) production and secretion of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2, producing effects comparable with those of the antimicrobial peptide LL-37. We also showed that the innate defense response mechanisms engaged by papiliocin involve Toll-like receptor pathways that culminate in the nuclear translocation of NF-κB. Fluorescent dye leakage experiments showed that papiliocin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of papiliocin in 300 mm dodecylphosphocholine micelles by NMR spectroscopy, showing that papiliocin has an α-helical structure from Lys(3) to Lys(21) and from Ala(25) to Val(36), linked by a hinge region. Interactions between the papiliocin and LPS studied using tryptophan blue-shift data, and saturation transfer difference-NMR experiments revealed that Trp(2) and Phe(5) at the N-terminal helix play an important role in attracting papiliocin to the cell membrane of Gram-negative bacteria. In conclusion, we have demonstrated that papiliocin is a potent peptide antibiotic with both anti-inflammatory and antibacterial activities, and we have laid the groundwork for future studies of its mechanism of action.     

The mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) and related phylogenetic analyses

The nearly complete mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) was sequenced for its nucleotide sequence of 13,964 bp. The genome has a typical gene order identical to other lepidopteran species. All tRNAs showed same stable canonical clover-leaf structure as those of other insects, except for tRNA^Ser (AGN), in which the dihydrouracil arm (DHU arm) could not form stable stem–loop structure. Anomalous initiation codons have been observed for the cox1 gene, where the ATTACG hexa-nucleotide was believed to be involved in the initiation signaling. Twelve mitochondrial protein-coding gene sequence data were used to infer the phylogenetic relationships among the insect orders. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the Homometabola was not supported in this paper. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, Papilionoidea and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea, Pyraloidea and Papilionoidea are monophyletic (Obtectomera). Bombycoidea (Bombyx mandarina and Antheraea pernyi) and Papilionoidea (P. xuthus and Coreana raphaelis) formed a sister group.     

Expression of one isoform of GTP cyclohydrolase I coincides with the larval black markings of the swallowtail butterfly, Papilio xuthus

The larva of the swallowtail butterfly Papilio xuthus changes its body markings during the fourth ecdysis. We found that stage-specific cuticular black markings are mainly regulated by co-localization of two melanin synthesis enzymes; tyrosine hydroxylase (TH) and dopa decarboxylase (DDC). TH converts tyrosine to dihydroxyphenylalanine (dopa), and tyrosine itself is converted from phenylalanine by phenylalanine hydroxylase (PAH). Guanosine triphosphate cyclohydrolase I (GTPCHI) is essential for the synthesis of tetrahydrobiopterin (BH4) that is a cofactor of TH and PAH. In this report, we found that a GTPCHI inhibitor prevents pigmentation in cultured integuments, suggesting that the GTPCHI activity is also involved in cuticle pigmentation. We have cloned GTPCHI and PAH cDNAs from P. xuthus and investigated their spatial expression patterns in epidermis by whole-mount in situ hybridization. There are two isoforms of GTPCHI in larval epidermis (GTPCHIa and GTPCHIb). GTPCHIa is expressed at the black markings of the subsequent instar, similar to TH, whereas GTPCHIb is expressed uniformly, similar to PAH. This suggests that the region-specific expression of GTPCHIa supplies sufficient BH(4) reinforcing the TH activity in black marking area. Our results imply that larval markings are regulated by not only melanin synthesis enzymes but also the cofactor supplying enzyme.