An anatomical and ultrastructural study of the eye of the luminescent millipede Paraspirobolus lucifugus (Gervais 1836) (Diplopoda, Spirobolida, Spiroboleliidae).

The two forward-looking eyes and their ultrastructural organization of an 18 mm long adult bioluminescent female millipede (Paraspirobolus lucifugus) were investigated by transmission electron microscopy and energy-dispersive X-ray spectroscopy. Each eye contained approximately 23 ommatidia with 50-60 µm wide and 80 um thick corneal lenses that contained calcium and silicon and proximally ended in truncated flat surfaces of around 20 µm in diameter. A maximally 28 µm thick and 25 µm long rhabdom, made up of at least 12-14 retinula cells and a 4 µm thick sleeve of screening pigment granules in a light-adapted position was present. Compared with the eyes of non-luminescent julid millipede species, those of P. lucifugus share their basic anatomy, but also exhibit features like the wide possible binocular frontal visual overlap, somewhat narrower interommatidial angles combined with relatively larger rhabdoms, which suggests that P. lucifugus has more efficient eyes and makes greater use of its photoreceptors. P. lucifugus is negatively phototactic and strictly nocturnal and its activity rhythm is apparently governed by a circadian clock.

Microscopy and biomimetics: the NanoSuit® method and image retrieval platform.

This review aims to clarify a suitable method towards achieving next-generation sustainability. As represented by the term 'Anthropocene', the Earth, including humans, is entering a critical era; therefore, science has a great responsibility to solve it. Biomimetics, the emulation of the models, systems and elements of nature, especially biological science, is a powerful tool to approach sustainability problems. Microscopy has made great progress with the technology of observing biological and artificial materials and its techniques have been continuously improved, most recently through the NanoSuit® method. As one of the most important tools across many facets of research and development, microscopy has produced a large amount of accumulated digital data. However, it is difficult to extract useful data for making things as biomimetic ideas despite a large amount of biological data. Here, we would like to find a way to organically connect the indispensable microscopic data with the new biomimetics to solve complex human problems.

Molecular Characterization and Tissue Distribution of Calcineurin Regulatory B Subunit during the Prevention of Diapause in the Silkworm, Bombyx mori.

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed calcineurin regulatory B subunit (CNB) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze CNB, which is known to associate with Ca2+. The gene expression level of CNB was increased by HCl treatment and the changes of the gene expression were almost the same as that in the non-diapause eggs. As for diapause eggs, almost no gene expression of CNB was confirmed except just after oviposition. In the assay for phosphorylation by protein kinase CK2, recombinant CNB (rCNB) was phosphorylated in vitro. Additionally, a Ca2+ binding assay indicated that rCNB shows affinity for Ca2+. The distribution of CNB was investigated with an immunohistochemical technique using antiserum against rCNB in diapause eggs and HCl-treated diapause eggs. CNB was localized in serosa cells and yolk cells in both eggs. These data may suggest that CNB is activated by intracellular Ca2+ or efflux Ca2+ resulting from HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.

Synergistic Effects of the Red Light and Blue Traps on Control of Thrips palmi (Thysanoptera: Thripidae).

The melon thrips (Thrips palmi) is a serious insect pest of vegetables and ornamental plants. To control and monitor this thrips, blue traps are widely used. We previously reported that irradiation of cucumber plants with red light-emitting diodes (660 nm) causes T. palmi to avoid the plants. Here, we evaluated the responses of T. palmi adults to a cucumber plant in an experimental arena with either a transparent sticky trap or a blue sticky trap, without or with illumination by a red LED panel. When T. palmi were introduced into the arena, the number of thrips attracted to the plant was lowest in treatments using the red light and blue trap. On the other hand, when T. palmi were inoculated on plants first, most thrips stayed put on the plants. Placing both red LEDs and blue sticky traps in greenhouses before the thrips invade the greenhouse from the outside might effectively control T. palmi.

Specializations in the compound eye of Talitrus saltator (Crustacea, Amphipoda).

We investigated the eye regionalization in Talitrus saltator by morphological, electrophysiological and behavioural experiments. Each ommatidium possesses five radially arranged retinular cells producing a square fused rhabdom by R1-R4 cells; the smaller R5 exists between R1 and R4. The size of R5 rhabdomere is larger in the dorsal part and becomes smaller in the median and ventral parts of the eye. Spectral-sensitivity by electroretinograms were recorded from dorsal or ventral parts of the eye. The dorsal part possesses maxima at green and UV-blue region. The main response region in the ventral part is only from UV (390 nm) to blue (430 nm) decreasing at longer wavelengths. To evaluate the sandhoppers' celestial orientation, their eyes were painted black either in the dorsal or ventral part, under the natural sky or a blue filter with or without the vision of the sun. Sandhoppers with the dorsal region of the eyes painted and tested under the screened sun were more dispersed and their directions varied more than in other groups of individuals. Sandhoppers with this area of the eye obscured display considerable difficulties to head in a specific direction. This work suggests the existence of regional specializations in the eye of T. saltator.

Framework with cytoskeletal actin filaments forming insect footpad hairs inspires biomimetic adhesive device design.

Footpads allow insects to walk on smooth surfaces. Specifically, liquid secretions on the footpad mediate adhesiveness through Van der Waals, Coulomb, and attractive capillary forces. Although the morphology and function of the footpad are well defined, the mechanism underlying their formation remains elusive. Here, we demonstrate that footpad hair in Drosophila is formed by the elongation of the hair cells and assembly of actin filaments. Knockdown of Actin5C caused a malformation of the hair structure, resulting in reduced ability to adhere to smooth substrates. We determined that functional footpads are created when hair cells form effective frameworks with actin filament bundles, thereby shaping the hair tip and facilitating cuticular deposition. We adapted this mechanism of microstructure formation to design a new artificial adhesive device⁠-a spatula-like fiber-framed adhesive device supported by nylon fibers with a gel material at the tip. This simple self-assembly mechanism facilitates the energy-efficient production of low-cost adhesion devices.

Molecular basis of wax-based color change and UV reflection in dragonflies.

Many animals change their body color for visual signaling and environmental adaptation. Some dragonflies show wax-based color change and ultraviolet (UV) reflection, but the biochemical properties underlying the phenomena are totally unknown. Here we investigated the UV-reflective abdominal wax of dragonflies, thereby identifying very long-chain methyl ketones and aldehydes as unique and major wax components. Little wax was detected on young adults, but dense wax secretion was found mainly on the dorsal abdomen of mature males of Orthetrum albistylum and O. melania, and pruinose wax secretion was identified on the ventral abdomen of mature females of O. albistylum and Sympetrum darwinianum. Comparative transcriptomics demonstrated drastic upregulation of the ELOVL17 gene, a member of the fatty acid elongase gene family, whose expression reflected the distribution of very long-chain methyl ketones. Synthetic 2-pentacosanone, the major component of dragonfly's wax, spontaneously formed light-scattering scale-like fine structures with strong UV reflection, suggesting its potential utility for biomimetics.

Molecular Characterization and Tissue Distribution of Mitochondrial Ca2+ -Dependent Solute Carrier Protein during Prevention of Diapause by HCl Treatment in the Silkworm, Bombyx mori.

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed mitochondrial Ca2+ -dependent solute carrier protein (MCSC) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze MCSC, which is known to associate with Ca2+ . The isolated cDNA of B. mori MCSC (BmMCSC) had an open reading flame (ORF) of 667 amino acid residues, and the ORF contained two EF-hand calcium-binding domains and three characteristic features of the mitochondrial solute carrier superfamily. The gene expression level of BmMCSC increased by HCl treatment. A Ca2+ binding assay indicated that recombinant BmMCSC (rBmMCSC) shows an affinity with Ca2 + . The distribution of BmMCSC was investigated with an immunohistochemical technique using antisera against BmMCSC in diapause eggs and HCl-treated diapause eggs. BmMCSC was localized in serosa cells in both eggs. These data may suggest that BmMCSC is activated by intracellular Ca2+ or efflux Ca2+ by HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.

A comparison between the larval eyes of the dimly luminescent Keroplatus nipponicus and the brightly luminescent Arachnocampa luminosa (Diptera; Keroplatidae).

Larvae of the weakly blue-luminescent fungus gnat Keroplatus nipponicus possess on either side of their heads a small black stemmatal eye with a plano-convex lens approximately 25 µm in diameter. In total, 12-14 retinula cells give rise to a centrally fused rhabdom of up to 8 µm in diameter. The rhabdom's constituent microvilli, approximately 70 nm in width, are roughly orthogonally oriented, a requirement for polarization sensitivity. Screening pigment granules are abundant in the retinula cells and measure at least 1 µm in diameter. In comparison with the stemmatal eye of the brightly luminescent Arachnocampa luminosa, that of K. nipponicus is considerably smaller with a poorer developed lens and a rhabdom that is less voluminous, but possesses wider microvilli. Although the larval eye of K. nipponicus can be expected to be functional, as the larvae react to light with a behavioural response, the eyes are probably mainly involved in the detection of ambient light levels and not, as in A. luminosa, also in responding to the luminescence of nearby conspecifics.

An RNAi Screen for Genes Involved in Nanoscale Protrusion Formation on Corneal Lens in Drosophila melanogaster.

The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions known as corneal nipples. Although the morphology and function of the "moth-eye" structure, are relatively well studied, the mechanism of protrusion formation from cell-secreted substances is unknown. In Drosophila melanogaster, a compound eye consists of approximately 800 facets, the surface of which is formed by the corneal lens with nanoscale protrusions. In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ßß subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila.

Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori.

Developmental changes in the localization of protein kinase CK2 in non-diapause and diapause eggs of the silkworm, Bombyx mori.

To analyze the role of protein kinase CK2 (CK2) during early embryogenesis in non-diapause and diapause of the silkworm, the distribution and localization of Bombyx mori CK2 (BmCK2) were investigated by an immunohistochemical technique using antibodies against the α- and ß-subunits of BmCK2. Both were localized in blastoderm cells of non-diapause and diapause eggs until 24 h after oviposition. More than 24 h after oviposition, however, the distribution of BmCK2 was different in non-diapause and diapause eggs. In non-diapause eggs, BmCK2 was mainly localized in yolk cells. In contrast, in diapause eggs, the localization was mainly observed in germ-band cells. Furthermore, we confirmed that the RNA helicase-like protein that was localized together with BmCK2 in non-diapause eggs was phosphorylated by BmCK2 in vitro. These data suggest that the role of BmCK2 is different in non-diapause and diapause eggs.

Ultrastructural analysis of lipid incorporation in the embryonic silkworm, Bombyx mori.

Insect eggs store many lipid droplets as an energy source for embryonic development. We previously reported that lipid droplets are incorporated into embryos in three steps in the silkworm, Bombyx mori. The midgut plays important roles in lipid incorporation during the second and third steps, whereas the manner of lipid incorporation during the first step is still unknown. In this study, we focused on how lipids were incorporated into the embryo in the first step, compared with the mechanisms used in the second step, by means of transmission electron microscopy using the high-pressure freezing and freeze substitution method. At the beginning of the first step (blastoderm formation stage), some lipid droplets were observed in each cell of the embryonic tissues. Lipid droplets were seen to be derived from the oocyte peripheral cytoplasm by superficial cleavage. At the end of the first step (late appendage formation stage), some lipid droplets were attached to the elongated rough endoplasmic reticulum (rER). It seemed that formation of the lipid droplets occurred in embryonic cells at the end of the first step, because the rER is the site of biogenesis of lipid droplets. The incorporation of lipid droplets in the first step may be subdivided into two stages: the blastoderm formation stage and the subsequent stage before blastokinesis.

Expression analysis of the aldo-keto reductases involved in the novel biosynthetic pathway of tetrahydrobiopterin in human and mouse tissues.

Tetrahydrobiopterin (BH(4)) acts as a cofactor of the aromatic amino-acid hydroxylases, and its deficiency may result in hyperphenylalaninemia (HPA) and decreased production of the neurotransmitters. BH(4) is synthesized by sepiapterin reductase (SPR) from 6-pyruvoyl-tetrahydropterin (PPH(4)). A patient with SPR deficiency shows no HPA; however, an SPR knockout mouse exhibits HPA. We have reported on the SPR-unrelated novel biosynthetic pathway from PPH(4) to BH(4) (salvage pathway II) in which 3alpha-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert. In this study, we performed the expression analysis of both proteins in humans and wild-type mice. The results of expression analysis indicated that salvage pathway II worked in human liver; however, it did not act in human brain or in mouse liver and brain. For this reason, a patient with SPR deficiency may show progressive neurological deterioration without HPA, and SPR knockout mice may exhibit HPA and abnormal locomotion activity.

Changes in lipid droplet localization during embryogenesis of the silkworm, Bombyx mori.

Lipid droplets are considered one of the most important energy sources in lepidopteran eggs during late embryogenesis, but the process of their incorporation into the embryo is as yet unknown. The present study focused on the process of transition of lipid droplets from the extraembryonic yolk to the embryo of the silkworm Bombyx mori, using morphological and biochemical approaches. The morphological study revealed that the incorporation of lipid droplets from the extraembryonic yolk into the embryo occurs at three points and in three different ways during the development of the embryo. Some lipid droplets were translocated directly from the extraembryonic yolk to the embryo before the blastokinesis stage. However, the majority of lipid droplets together with the other components of the extraembryonic yolk were incorporated in the embryo via both morphogenetic inclusion during dorsal closure and ingestion of the extraembyonic yolk by the developing caterpillar prior to hatching. Similar results were obtained from the biochemical study. Thus, we propose that there are three steps in the incorporation of lipid droplets from the extraembryonic yolk into the embryo. In addition, morphological and biochemical data concerning the total amount of lipid droplets in the egg suggested that lipid droplets were mainly consumed during late embryogenesis, seeming to synchronize with tracheal development.

Presence of rhodopsin and porphyropsin in the eyes of 164 fishes, representing marine, diadromous, coastal and freshwater species--a qualitative and comparative study.

There are two types of visual pigments in fish eyes; most marine fishes have rhodopsin, while most freshwater fishes have porphyropsin. The biochemical basis for this dichotomy is the nature of the chromophores, retinal (A1) and 3-dehydroretinal (A2), each of which is bound by an opsin. In order to study the regional distribution of these visual pigments, we performed a new survey of the visual pigment chromophores in the eyes of many species of fish. Fish eyes from 164 species were used to examine their chromophores by high-performance liquid chromatography--44 species of freshwater fish, 20 of peripheral freshwater fish (coastal species), 10 of diadromous fish and 90 of seawater fish (marine species) were studied. The eyes of freshwater fish, limb freshwater fish and diadromous fish had both A1 and A2 chromophores, whereas those of marine fish possessed only A1 chromophores. Our results are similar to those of previous studies; however, we made a new finding that fish which live in freshwater possessed A1 if living near the sea and A2 if living far from the sea if they possessed only one type of chromophore.

Molecular properties and tissue distribution of 30K proteins as ommin-binding proteins from diapause eggs of the silkworm, Bombyx mori.

We previously reported the purification of an ommin-binding protein (OMBP) from an acid-methanol extract of diapause eggs of the silkworm and that OMBP reacted with the anti-30K proteins antiserum. In order to clarify the relationship between OMBP and the 30K proteins, we attempted to determine the sequence of the N-terminal amino acid of OMBP, which was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We observed ten protein spots of various isoelectric points; the spots corresponded with 30 kDa. Based on the sequence of the N-terminal amino acid (20 residues), the spots belonged to two kinds of 30K proteins (6G1 and 19G1), which are known as the major plasma proteins in the larval hemolymph of the silkworm. The proteins are expected to attach to polysaccharide because they reacted with concanavalin A and elderberry bark lectin. Immunohistochemical observations clarified that the proteins were localized in yolk granules and serosa in the diapause egg. These results suggest that OMBP is composed of 30K proteins which were modified with polysaccharides. In addition, the expression of 30K proteins mRNA was observed at early embryonic stage in diapause eggs by RT-PCR analysis. The 30K proteins as OMBP may play an important role in the transport and accumulation of tryptophan metabolites and ommochrome during the formation of serosa.

Release of ecdysteroid-phosphates from egg yolk granules and their dephosphorylation during early embryonic development in silkworm, Bombyx mori.

Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.

Egg transport in the coelom of the newt cynops pyrrhogaster. I. The ovulated egg is transported to the ostium by the ciliary movement of coelomic epithelial cells.

We investigated the mechanism of egg transport in the newt not only by inserting various conditioned eggs into the recipient's body but also by placing them on the coelomic epithelia of the opened body cavity in the adult female newt. Most of the inserted coelomic eggs were oviposited, while 4 of 14 inserted de-jellied uterine eggs and 3 of 10 inserted de-jellied fertilized eggs were oviposited. The coelomic eggs placed on the coelomic epithelia were transported toward the ostium and entered the ostium. The de-jellied uterine eggs and the de-jellied fertilized eggs were transported to the ostium as well. Of all the eggs examined, the coelomic egg was transported the fastest. The transport speeds of coelomic eggs treated with periodic acid and the speed of boiled coelomic eggs were less than those of untreated coelomic eggs. In contrast, the transport speeds of coelomic eggs treated with trypsin and the speed of coelomic eggs removed from their vitelline envelopes (naked eggs) were faster than those of untreated coelomic eggs. Other experiments were carried out in order to ascertain the dependence of sexual activity on egg transport. The speed of coelomic egg transport in artificially sexually activated females was faster than in sexually inactive females, although the ciliary movement could always be observed in both sexually active females and sexually inactive females. This suggests that the speed of egg transport on the coelomic epithelia is controlled by the sexual activity of the female.