Functional unit of supergene in female-limited Batesian mimicry of Papilio polytes.

Supergenes are sets of genes and genetic elements that are inherited like a single gene and control complex adaptive traits, but their functional roles and units are poorly understood. In Papilio polytes, female-limited Batesian mimicry is thought to be regulated by a ∼130 kb inversion region (highly diversified region: HDR) containing 3 genes, UXT, U3X, and doublesex (dsx) which switches non-mimetic and mimetic types. To determine the functional unit, we here performed electroporation-mediated RNAi analyses (and further Crispr/Cas9 for UXT) of genes within and flanking the HDR in pupal hindwings. We first clarified that non-mimetic dsx-h had a function to form the non-mimetic trait in female and only dsx-H isoform 3 had an important function in the formation of mimetic traits. Next, we found that UXT was involved in making mimetic-type pale-yellow spots and adjacent gene sir2 in making red spots in hindwings, both of which refine more elaborate mimicry. Furthermore, downstream gene networks of dsx, U3X, and UXT screened by RNA sequencing showed that U3X upregulated dsx-H expression and repressed UXT expression. These findings demonstrate that a set of multiple genes, not only inside but also flanking HDR, can function as supergene members, which extends the definition of supergene unit than we considered before. Also, our results indicate that dsx functions as the switching gene and some other genes such as UXT and sir2 within the supergene unit work as the modifier gene.

Genetic switch in UV response of mimicry-related pale-yellow colors in Batesian mimic butterfly, Papilio polytes.

In a Batesian mimic butterfly Papilio polytes, mimetic females resemble an unpalatable model, Pachliopta aristolochiae, but exhibit a different color pattern from nonmimetic females and males. In particular, the pale-yellow region on hind wings, which correspondingly sends important putative signals for mimicry and mate preference, is different in shape and chemical features between nonmimetic and mimetic morphs. Recently, we found that mimetic-type doublesex [dsx (H)] causes mimetic traits; however, the control of dimorphic pale-yellow colors remains unclear. Here, we revealed that dsx (H) switched the pale-yellow colors from UV-excited fluorescent type (nonmimetic) to UV-reflecting type (mimetic), by repressing the papiliochrome II synthesis genes and nanostructural changes in wing scales. Photoreceptor reactivities showed that some birds and butterflies could effectively recognize mimetic and nonmimetic pale-yellow colors, suggesting that a genetic switch in the UV response of pale-yellow colors may play essential roles in establishing the dimorphic female-limited Batesian mimicry.

Notch and Delta Control the Switch and Formation of Camouflage Patterns in Caterpillars.

In most Papilio species, a younger larva mimics bird droppings but changes its pattern to match host plant colors in its final instar. This change is determined by juvenile hormone (JH) during the JH-sensitive period (JHSP) early in the fourth instar. Recently, we found that homeobox genes control the pre-pattern formation specifically during JHSP, but the molecular mechanisms underlying final patterning and pigmentation at molt are unknown. By knockdown of Delta and Notch in Papilio xuthus larvae, we here showed that these genes define the edge and pigmentation area in final patterns, during and even after JHSP, suggesting that they bridge the JHSP and molt. Knockdown of Delta in Papilio machaon led to similar phenotypic changes, and knockdown of Notch caused pigmentation loss in twin spots of the silkworm Multilunar (L) mutant. Our findings suggest the importance of the Notch signaling pathway in caterpillars' adaptive evolution of color pattern formation.

The mimetic wing pattern of Papilio polytes butterflies is regulated by a doublesex-orchestrated gene network.

The swallowtail butterfly Papilio polytes is sexually dimorphic and exhibits female-limited Batesian mimicry. This species also has two female forms, a non-mimetic form with male-like wing patterns, and a mimetic form resembling an unpalatable model, Pachliopta aristolochiae. The mimicry locus H constitutes a dimorphic Mendelian 'supergene', including a transcription factor gene doublesex (dsx). However, how the mimetic-type dsx (dsx-H) orchestrates the downstream gene network and causes the mimetic traits remains unclear. Here we performed RNA-seq-based gene screening and found that Wnt1 and Wnt6 are up-regulated by dsx-H during the early pupal stage and are involved in the red/white pigmentation and patterning of mimetic female wings. In contrast, a homeobox gene abdominal-A is repressed by dsx-H and involved in the non-mimetic colouration pattern. These findings suggest that dual regulation by dsx-H, induction of mimetic gene networks and repression of non-mimetic gene networks, is essential for the switch from non-mimetic to mimetic pattern in mimetic female wings.

Toll ligand Spätzle3 controls melanization in the stripe pattern formation in caterpillars.

A stripe pattern is an aposematic or camouflage coloration often observed among various caterpillars. However, how this ecologically important pattern is formed is largely unknown. The silkworm dominant mutant Zebra (Ze) has a black stripe in the anterior margin of each dorsal segment. Here, fine linkage mapping of 3,135 larvae revealed a 63-kbp region responsible for the Ze locus, which contained three candidate genes, including the Toll ligand gene spätzle3 (spz-3). Both electroporation-mediated ectopic expression and RNAi analyses showed that, among candidate genes, only processed spz-3 induced melanin pigmentation and that Toll-8 was the candidate receptor gene of spz-3 This Toll ligand/receptor set is also involved in melanization of other mutant Striped (pS ), which has broader stripes. Additional knockdown of 5 other spz family and 10 Toll-related genes caused no drastic change in the pigmentation of either mutant, suggesting that only spz-3/Toll-8 is mainly involved in the melanization process rather than pattern formation. The downstream pigmentation gene yellow was specifically up-regulated in the striped region of the Ze mutant, but spz-3 showed no such region-specific expression. Toll signaling pathways are known to be involved in innate immunity, dorsoventral axis formation, and neurotrophic functions. This study provides direct evidence that a Toll signaling pathway is co-opted to control the melanization process and adaptive striped pattern formation in caterpillars.

Aerodynamic levitator for large-sized glassy material production.

Containerless aerodynamic levitation processing is a unique technology for the fabrication of bulk non-crystalline materials. Using conventional aerodynamic levitation, a high reflective index (RI) material (BaTi2O5 and LaO3/2-TiO2-ZrO2 system) was developed with a RI greater than approximately 2.2, which is similar to that of diamond. However, the glass size was small, approximately 3 mm in diameter. Therefore, it is essential to produce large sized materials for future optical materials applications, such as camera lenses. In this study, a new aerodynamic levitator was designed to produce non-crystalline materials with diameters larger than 6 mm. The concept of this new levitator was to set up a reduced pressure at the top of the molten samples without generating turbulent flow. A numerical simulation was also performed to verify the concept.

The transcription factor Apontic-like controls diverse colouration pattern in caterpillars.

Genetic polymorphisms underlie the convergent and divergent evolution of various phenotypes. Diverse colour patterns on caterpillars, which are ecologically important, are good models for understanding the molecular backgrounds of phenotypic diversity. Here we show that a single evolutionarily conserved gene apontic-like (apt-like) encoding for a putative transcription factor accounts for the silkworm p locus, which causes at least 15 different larval markings involved in branch-like markings and eye-spot formation. The expression of apt-like and melanin synthesis genes are upregulated in association with pigmented areas of marking mutants Striped (p(S)) and normal (+(p)) but not in the non-marking allele plain (p). Functional analyses, ectopic expression, RNAi and TALEN, demonstrate that apt-like causes melanin pigmentation in a cell-autonomous manner. These results suggest that variation in p alleles is caused by the differential expression of the gene apt-like which induces targeted elevation of gene expressions in the melanin synthesis pathway.

The TUNEL assay suggests mandibular regression by programmed cell death during presoldier differentiation in the nasute termite Nasutitermes takasagoensis.

Termite soldiers are the most specialized caste of social insects in terms of their morphology and function. Soldier development requires increased juvenile hormone (JH) titer and the two molts via a presoldier stage. These molts are accompanied by dramatic morphological changes, including the exaggeration and regression of certain organs. Soldiers of the most apical termitid subfamily Nasutitermitinae possess not only a horn-like frontal tube, called the nasus, for the projection of defensive chemicals from the frontal gland reservoir but also regressed mandibles. Although candidate genes regulating soldier mandibular growth were reported in a relatively basal termite species, the regulatory mechanisms of mandibular regression remain unknown. To clarify these mechanisms, we performed morphological and histological examinations of the mandibles during soldier differentiation in Nasutitermes takasagoensis. Mandibular size reduced dramatically during soldier differentiation, and mandibular regression occurred just prior to the presoldier molt. Spotted TUNEL signals were observed in regressing mandibles of presoldiers, suggesting that the regression involved programmed cell death. Because soldiers of N. takasagoensis possess exaggerated organs (nasus and frontal gland), the present results suggest that JH-dependent regressive mechanisms exist in the mandibles without interfering with the formation of the exaggerated organs.

Compact electrostatic levitator for diffraction measurements with a two axis diffractometer and a laboratory x-ray source.

A compact electrostatic levitator was developed for the structural analysis of high-temperature liquids by x-ray diffraction methods. The size of the levitator was 200 mm in diameter and 200 mm in height and can be set up on a two axis diffractometer with a laboratory x-ray source, which is very convenient in performing structural measurements of high-temperature liquids. In particular, since the laboratory x-ray source allows a great amount of user time, preliminary or challenging experiments can be performed with trial and error, which prepares and complements synchrotron x-ray experiments. The present small apparatus also provides the advantage of portability and facility of setting. To demonstrate the capability of this electrostatic levitator, the static structure factors of alumina and silicon samples in their liquid phases were successfully measured.

Convection effects on crystallinity in the growth of In0.3Ga0.7 as crystals by the traveling liquidus zone method.

The influence of convection in a melt on the crystallinity of the TLZ-grown In(0.3)Ga(0.7)As crystals has been investigated by growing crystals with various shapes and dimensions on the ground. No single crystals have been grown when the crystal diameter was 10 mm, but we were successful in growing single crystals by reducing crystal diameter to 2 mm. These results suggested the importance of suppressing convection in the melt during alloy crystal growth because constitutional supercooling tends to occur at the freezing interface or ahead of the interface by the segregation effect. Large area is required for substrate use in various applications. This requirement can be fulfilled by the crystal growth in microgravity because density difference-induced convection is suppressed in microgravity. Another means for suppressing convection without deteriorating area is plate-shape crystal growth with reduced thickness. The latter can be applied on the ground and we succeeded in growing single crystals of plate-shaped In(0.3)Ga(0.7)As by the traveling liquidus zone (TLZ) method. Dimensions of obtained single crystals were 10 mm in width and 2 mm in thickness and lengths ranged from 20 to 40 mm. Compositional uniformity was good and 0.3 +/- 0.02 in InAs mole fraction was achieved.

Impurity effects on lysozyme crystal growth.

Atomic force microscopy (AFM) and X-ray diffraction experiments have been combined to study the correlation between impurity incorporation, crystal surface morphology and crystal quality. Hen egg-white lysozyme has been used as a model protein, and covalently bound lysozyme dimer as a model impurity. AFM observation of the [101] crystal face revealed that the crystal surface clearly became rough when 5% impurity was added, and the steps disappeared as the impurity concentration increased to 10%. The crystal quality was evaluated by four factors: maximum resolution limit, /, Rmerge, and overall B factor. In every index, the crystal quality tended to degrade as the impurity concentration increased. The B-factor dropped significantly at 5% impurity; at the same time the step roughening was observed. This strongly suggested that the impurity incorporation affected the step growth mechanism and degraded the crystal quality.

Improvement of protein crystal quality by forced flow solution.

Flow experiments in growing protein crystals were conducted to clarify the influence of the solution flow on the crystal quality. Lysozyme crystals grown under various flow velocities were analyzed by using synchrotron radiation to assess the quality. As a result, the crystals grown under forced flow were of better quality than those grown in quiescent conditions.

Scientific approach to the optimization of protein crystallization conditions for microgravity experiments.

The National Space Development Agency of Japan (NASDA) developed a practical protocol to optimize protein crystallization conditions for microgravity experiments. This protocol focuses on the vapor diffusion method using high density protein crystal growth (HDPCG)--hardware developed by the University of Alabama, Birmingham--that flew on the STS-107 mission. The objective of this development was to increase the success rate of microgravity experiments by setting crystallization conditions based on knowledge of crystal growth and fluid dynamics. The protocol consists of four steps: (1) phase diagram preparation, (2) estimation of condensation rate in the vapor diffusion method, (3) fluid dynamic property measurement, and (4) fluid dynamic simulation. First, a phase diagram was constructed. Crystallization characteristics were investigated by a microbatch method. The data were recalculated based on classical nucleation theory and the crystallization boundary was determined as a function of time. The second step was to develop a practical model to estimate the condensation rate of the crystallizing solution, including protein and precipitant, as a function of the precipitant concentration and solution volume. By considering the crystallization map and the vapor diffusion condensation model we were able to optimize the crystallization conditions that generate crystals in the desired time. This was particularly important in a shuttle mission whose mission duration is limited. The third step was fluid dynamic property measurement necessary for fluid dynamics simulation and crystal growth study. The last step was to estimate the mass transport in space on the basis of the fluid dynamics simulation transport model. It turned out that neither the vapor phase nor the solution phase was seriously affected by gravity until nucleation provided the hardware was set in a normal direction. Therefore, we concluded that the optimized crystallization conditions could be directly applied to microgravity experiments. By completing the approach, we were able to control the time for nucleation in the vapor diffusion method.

Property measurements and solidification studies by electrostatic levitation.

The National Space Development Agency of Japan has recently developed several electrostatic levitation furnaces and implemented new techniques and procedures for property measurement, solidification studies, and atomic structure research. In addition to the contamination-free environment for undercooled and liquid metals and semiconductors, the newly developed facilities possess the unique capabilities of handling ceramics and high vapor pressure materials, reducing processing time, and imaging high luminosity samples. These are exemplified in this paper with the successful processing of BaTiO(3). This allowed measurement of the density of high temperature solid, liquid, and undercooled phases. Furthermore, the material resulting from containerless solidification consisted of micrometer-size particles and a glass-like phase exhibiting a giant dielectric constant exceeding 100,000.