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Angelman syndrome is a neurological disorder whose symptoms include severe mental retardation, loss of motor coordination, and sleep disturbances. The disease is caused by a loss of function of UBE3A, which encodes a HECT-domain ubiquitin ligase. Here, we generate a Drosophila model for the disease. The results of several experiments show that the functions of human UBE3A and its fly counterpart, dube3a, are similar. First, expression of Dube3a is enriched in the Drosophila nervous system, including mushroom bodies, the seat of learning and memory. Second, we have generated dube3a null mutants, and they appear normal externally, but display abnormal locomotive behavior and circadian rhythms, and defective long-term memory. Third, flies that overexpress Dube3a in the nervous system also display locomotion defects, dependent on the ubiquitin ligase activity. Finally, missense mutations in UBE3A alleles of Angelman syndrome patients alter amino acid residues conserved in the fly protein, and when introduced into dube3a, behave as loss-of-function mutations. The simplest model for Angelman syndrome is that in the absence of UBE3A, particular substrates fail to be ubiquitinated and proteasomally degraded, accumulate in the brain, and interfere with brain function. We have generated flies useful for genetic screens to identify Dube3a substrates. These flies overexpress Dube3a in the eye or wing and display morphological abnormalities, dependent on the critical catalytic cysteine. We conclude that dube3a mutants are a valid model for Angelman syndrome, with great potential for identifying the elusive UBE3A substrates relevant to the disease.

Omnivorous animals feed on several food items that often differ in macronutrient and isotopic composition. Macronutrients can be used for either metabolism or body tissue synthesis and, therefore, stable C isotope ratios of exhaled breath (delta(13)C(breath)) and tissue may differ. To study nutrient routing in omnivorous animals, we measured delta(13)C(breath) in 20-g Carollia perspicillata that either ate an isotopically homogeneous carbohydrate diet or an isotopically heterogeneous protein-carbohydrate mixture. The delta(13)C(breath) converged to the delta(13)C of the ingested carbohydrates irrespective of whether proteins had been added or not. On average, delta(13)C(breath) was depleted in (13)C by only ca. -2 per thousand in relation to the delta(13)C of the dietary carbohydrates and was enriched by +8.2 per thousand in relation to the dietary proteins, suggesting that C. perspicillata may have routed most ingested proteins to body synthesis and not to metabolism. We next compared the delta(13)C(breath) with that of wing tissue (delta(13)C(tissue)) in 12 free-ranging, mostly omnivorous phyllostomid bat species. We predicted that species with a more insect biased diet--as indicated by the N isotope ratio in wing membrane tissue (delta(15)N(tissue))--should have higher delta(13)C(tissue) than delta(13)C(breath) values, since we expected body tissue to stem mostly from insect proteins and exhaled CO(2) to stem from the combustion of fruit carbohydrates. Accordingly, delta(13)C(tissue) and delta(13)C(breath) should be more similar in species that feed predominantly on plant products. The species-specific differences between delta(13)C(tissue) and delta(13)C(breath) increased with increasing delta(15)N(tissue), i.e. species with a plant-dominated diet had similar delta(13)C(tissue) and delta(13)C(breath) values, whereas species feeding at a higher trophic level had higher delta(13)C(tissue) than delta(13)C(breath) values. Our study shows that delta(13)C(breath) reflect the isotope ratio of ingested carbohydrates, whereas delta(13)C of body tissue reflect the isotope ratio of ingested proteins, namely insects, supporting the idea of isotopic routing in omnivorous animals.

A mechanistic understanding of the butterfly wing color-pattern determination can be facilitated by experimental pattern changes. Here I review physiologically induced color-pattern changes in nymphalid butterflies and their mechanistic and evolutionary implications. A type of color-pattern change can be elicited by elemental changes in size and position throughout the wing, as suggested by the nymphalid groundplan. These changes of pattern elements are bi-directional and bi-sided dislocation toward or away from eyespot foci and in both proximal and distal sides of the foci. The peripheral elements are dislocated even in the eyespot-less compartments. Anterior spots are more severely modified, suggesting the existence of an anterior-posterior gradient. In one species, eyespots are transformed into white spots with remnant-like orange scales, and such patterns emerge even at the eyespot-less "imaginary" foci. A series of these color-pattern modifications probably reveal "snap-shots" of a dynamic morphogenic signal due to heterochronic uncoupling between the signaling and reception steps. The conventional gradient model can be revised to account for these observed color-pattern changes.

Formation and function of the A/P compartment boundary of the Drosophila wing have been studied intensely. The boundary prevents mingling of A and P cells, is characterized by an expression discontinuity of several genes like engrailed, Cubitus interruptus, hedgehog and decapentaplegic and is essential for patterning the wing. Compared with segmental or compartmental boundaries in several other systems which generally manifest as folds or clefts, the wing A/P boundary is morphologically inconspicuous in both the larval and adult stage. We show here that the Drosophila wing A/P boundary, too, is susceptible to fold and cleft formation and that these processes are suppressed by the T-box transcription factor Optomotor-blind (Omb). Using a targeted deletion encompassing the omb wing enhancer [Sivasankaran, R., Vigano, M.A., Muller, B., Affolter, M., Basler, K., 2000. Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker. EMBO J. 19, 6162-6172] and omb-RNAi to strongly and specifically reduce Omb, we show that omb is required in P cells to prevent aberrant apical fold formation at the A/P boundary of the larval wing disc and cleft formation in the adult wing blade. In the larval A/P fold, DE-cadherin-based adherens junctions appeared intact but the apical microtubule web was strongly reduced.

Male crickets display sex-specific (e.g., mating and agonistic) behaviors towards conspecific individuals. One of the key signals for these behaviors is the chemical substance on the cricket body surface. In the present study, we analyzed female and male cuticular substances in behavioral assays. Antennal contact stimulation using female forewings elicited a mating behavior in males, while that using male forewings elicited an agonistic behavior in males. Thin-layer-chromatographic and other techniques analysis showed that saturated cuticular lipids were present in both female and male cuticles and that unsaturated lipids were present only in the male cuticle. Filter papers soaked with saturated or unsaturated cuticular lipids were applied to antennae of male crickets. Males showed mating behavior in response to stimulation with saturated lipids from both females and males but showed avoidance behavior in response to stimulation with male unsaturated lipids. Because cuticular lipids did not induce agonistic behavior in males, we collected odors from male crickets and found that these odors induced agonistic behavior in males. Therefore, we concluded that the key signals for mating, avoidance and agonistic behaviors of male crickets are comprised of at least three different components, saturated and unsaturated cuticular lipids and male odors, respectively.

The stable subdivision of Drosophila limbs into anterior and posterior compartments is a consequence of asymmetrical signalling by Hedgehog (Hh), from the posterior to anterior cells. The activity of the homeodomain protein Engrailed in posterior cells helps to generate this asymmetry by inducing the expression of Hh in the posterior compartment and, at the same time, repressing the expression of the essential downstream component Cubitus interruptus (Ci). Therefore, only anterior cells that receive the Hh signal across the compartment boundary will respond by stabilizing Ci. Here, we describe a new molecular mechanism that helps to maintain the Hh-expressing and Hh-responding cells in different non-overlapping cell populations. Master of thickveins (mtv) - a target of Hh activity encoding a nuclear zinc-finger protein - is required to repress hh expression in anterior cells. Mtv exerts this action in a protein complex with Groucho (Gro) - the founding member of a superfamily of transcriptional corepressors that are conserved throughout eukaryotes. Therefore, Hh restricts its own expression domain in the Drosophila wing through the activity of Mtv and Gro.

In 1997 and 1998 we weighed nestling tree swallows (Tachycineta bicolor) and measured selected body components at two colonies: Acadia National Park on Mt. Desert Island, and at Orono, ME. We used differences in mean growth variables among individual nestlings to evaluate differences between colonies, years, and amount of total mercury (THg) in carcasses and methyl mercury (MeHg) in feathers. We marked nestlings on the day hatched and measured body components every day in 1997 and every other day in 1998 until nestlings fledged. We calculated linear growth rates and asymptotic means as appropriate. In 1998, linear growth rate of weight was higher at Acadia than at Orono, but not different in 1997. We detected no mean differences in asymptotic mean weight of nestlings between colonies or years. In 1997, mean linear growth rates of the wing (chord), tail, tarsus, and mandible were higher at Acadia than at Orono. The amount of MeHg in feathers was associated with a lower linear growth rate of weight during early age (2-10 days), but asymptotic mean weight during days 11-16 was not different. No effect on linear growth of tail feathers or wing was associated with the amount of MeHg in feathers or THg in carcasses. Fledgling tree swallows that survive to migrate, however, will leave Maine with substantial concentrations of Hg in their tissues.

The beads in the wing scales of pierid butterflies play a crucially important role in wing coloration as shown by spectrophotometry and scanning electron microscopy (SEM). The beads contain pterin pigments, which in Pieris rapae absorb predominantly in the ultraviolet (UV). SEM demonstrates that in the European subspecies Pieris rapae rapae, both males and females have dorsal wing scales with a high concentration of beads. In the Japanese subspecies Pieris rapae crucivora, however, only the males have dorsal wing scales studded with beads, and the dorsal scales of females lack beads. Microspectrophotometry of single scales without beads yields reflectance spectra that increase slightly and monotonically with wavelength. With beads, the reflectance is strongly reduced in the UV and enhanced at the longer wavelengths. By stacking several layers of beaded scales, pierid butterflies achieve strong colour contrasts, which are not realized in the dorsal wings of female P. r. crucivora. Consequently, P. r. crucivora exhibits a strong sexual dichroism that is absent in P. r. rapae.

As organisms develop, their tissues can become separated into distinct cell populations through the establishment of compartment boundaries. Compartment boundaries have been discovered in a wide variety of tissues, but in many cases the molecular mechanisms that separate cells remain poorly understood. In the Drosophila wing, a stripe of Notch activation maintains the dorsal-ventral compartment boundary, through a process that depends on the actin cytoskeleton. Here, we show that the dorsal-ventral boundary exhibits a distinct accumulation of Myosin II, and that this accumulation is regulated downstream of Notch signaling. Conversely, the dorsal-ventral boundary is depleted for the Par-3 homologue Bazooka. We further show that mutations in the Myosin heavy chain subunit encoded by zipper can impair dorsal-ventral compartmentalization without affecting anterior-posterior compartmentalization. These observations identify a distinct accumulation and requirement for Myosin activity in dorsal-ventral compartmentalization, and suggest a novel mechanism in which contractile tension along an F-actin cable at the compartment boundary contributes to compartmentalization., ((c) 2006 Wiley-Liss, Inc.)

Developmentally regulated programmed cell death sculpts the limbs and other embryonic organs in vertebrates. One intriguing example of species-specific differences in apoptotic extent is observed in the tissue between the digits. In chicks and mice, bone morphogenetic proteins (Bmps) trigger apoptosis of the interdigital mesenchyme, leading to freed digits, whereas in ducks, Bmp antagonists inhibit the apoptotic program, resulting in webbed feet. Here, we show that the phyllostomid bat Carollia perspicillata utilizes a distinct mechanism for maintaining interdigit tissue. We find that bat forelimb and hindlimb interdigital tissues express Bmp signaling components but that only bat hindlimbs undergo interdigital apoptosis. Strikingly, the retention of interdigital webbing in the bat forelimb is correlated with a unique pattern of Fgf8 expression in addition to the Bmp inhibitor Gremlin. By using a functional assay, we show that maintenance of interdigit tissue in the bat wing depends on the combined effects of high levels of Fgf signaling and inhibition of Bmp signaling. Our data also indicate that although there is not a conserved mechanism for maintaining interdigit tissue across amniotes, the expression in the bat forelimb interdigits of Gremlin and Fgf8 suggests that these key molecular changes contributed to the evolution of the bat wing.

The fine structure of the wing scale of a Morpho Peleides butterfly was examined carefully, and the entire configuration was completely replicated by a uniform Al(2)O(3) coating through a low-temperature ALD process. An inverted structure was achieved by removing the butterfly wing template at high temperature, forming a polycrystalline Al(2)O(3) shell structure with precisely controlled thickness. Other than the copy of the morphology of the structure, the optical property, such as the existence of PBG, was also inherited by the alumina replica. Reflection peaks at the violet/blue range were detected on both original wings and their replica, while a simple alumina coating shifted the reflection peak to longer wavelength because of the change of periodicity and refraction index. The alumina replicas also exhibited similar functional structures as waveguide and beam splitter, which may be used as the building blocks for photonic ICs with high reproducibility and lower fabrication cost compared to traditional lithography techniques.

The size and shape of organs depend on cellular processes such as cell proliferation, cell survival, and spatial arrangement of cells. In turn, all of these processes are a consequence of positional identity of individual cells in whole organs. Links of positional information with organ growth and pattern expression of genes is a little-addressed question. We show that differences in vestigial expression between neighboring cells of the wing blade autonomously and nonautonomously affect cell proliferation along the proximo-distal axis. On the other hand, uniform expression of vestigial inhibits cell proliferation and also perturbs the shape of wing blade altering the preferential orientation of cell divisions. Our observations provide evidence that local cell interactions, triggered by differences in vestigial expression between neighboring cells, confer positional values operating in the control of growth and shape of the wing.

Nano-structured colorful zinc oxide (ZnO) replicas were produced using the wings of the Ideopsis similis butterfly as templates. The ZnO replicas we obtained exhibit iridescence, which was clearly observed under an optical microscope (OM). Field emission scanning electron microscope analysis shows that all the microstructure details are maintained faithfully in the ZnO replica. A computer model was established to simulate the diffraction spectral results, which agreed well with the OM images.

Photonic-crystal-type nanostructures occurring in the scales of the butterfly Cyanophrys remus were investigated by optical and electron microscopy (scanning and transmission electron microscopy), reflectance measurements (specular, integrated, and goniometric), by fast Fourier transform analysis of micrographs, by modeling, and by numerical simulation of the measured reflectance data. By evaluating the collected data in a cross-correlated way, we show that the metallic blue dorsal coloration originates from scales which individually are photonic single crystals of 50 x 120 microm2 , while the matt pea-green coloration of the ventral side arises from the cumulative effect of randomly arranged, bright photonic crystallites (blue, green, and yellow) with typical diameters in the 3-10-mum range. Both structures are based on a very moderate refractive index contrast between air and chitin. Using a bleached specimen in which the pigment has decayed with time, we investigated the role of pigment in photonic-crystal material in the process of color generation. The possible biologic utility of the metallic blue (single-crystal) and dull green (polycrystal) textures both achieved with photonic crystals are briefly discussed. Potential applications in the field of colorants, flat panel displays, smart textiles, and smart papers are surveyed.

E75A and E75B, isoforms of the E75 orphan nuclear receptor, are sequentially up-regulated in the abdominal epidermis of the tobacco hornworm Manduca sexta by 20-hydroxyecdysone (20E) during larval and pupal molts, with E75A also increasing at pupal commitment (Zhou et al., Dev. Biol. 193, 127-138, 1998). We have now cloned E75C and show that little is expressed in the epidermis during larval life with trace amounts seen just before ecdysis. Instead, E75C is found in high amounts during the development of the adult wings as the ecdysteroid titer is rising, and this increase was prevented by juvenile hormone (JH) that prevented adult development. By contrast, E75D is expressed transiently during the larval and pupal molts as the ecdysteroid titer begins to decline and again just before ecdysis, but in the developing adult wings is expressed on the rise of 20E. Removal of the source of JH had little effect on either E75C or E75D mRNA expression during the larval and pupal molts. At the time of pupal commitment, in vitro experiments show that 20E up-regulates E75D and JH prevents this increase. Neither E75A nor E75D mRNA was up-regulated by JH alone. Thus, E75C is primarily involved in adult differentiation whereas E75D has roles both during the molt and pupal commitment.

The present study demonstrates a surface structure that mimics the water harvesting wing surface of the Namib Desert beetle. Hydrophilic patterns on superhydrophobic surfaces were created with water/2-propanol solutions of a polyelectrolyte to produce surfaces with extreme hydrophobic contrast. Selective deposition of multilayer films onto the hydrophilic patterns introduces different properties to the area including superhydrophilicity. Potential applications of such surfaces include water harvesting surfaces, controlled drug release coatings, open-air microchannel devices, and lab-on-chip devices.

In this paper we showed a new approach for the fabrication of a photonic crystal with a three-dimensional structure. By replicating biomaterials such as the wing of Mopho butterfly with TiO2 nanoparticles using the nanoparticles infiltration method, we can derive photonic crystals with unique structures, which is difficult to fabricate by other approaches. New optical properties are anticipated.

Biomimetic research indicates that many phenomena regarding wettability in nature, such as the self-cleaning effect on a lotus leaf and cicada wing, the anisotropic dewetting behavior on a rice leaf, and striking superhydrophobic force provided by a water strider's leg, are all related to the unique micro- and nanostructures on the surfaces. It gives us much inspiration to realize special wettability on functional surfaces through the cooperation between the chemical composition and the surface micro- and nanostructures, which may bring great advantages in a wide variety of applications in daily life, industry, and agriculture. This Account reviews recent progress in these aspects.

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with beta-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed beta-D-glucuronic acid and N-acetyl beta-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

We have combined high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs of Drosophila melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S]methionine + [35S]cysteine-labeled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin, and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies reveal qualitative and quantitative differences between the two samples, but have more than 600 polypeptides in common. Second, we carried out preparative 2-D polyacrylamide gel electrophoresis using Schneider cells mixed with radioactively labeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them by matrix-assisted laser desorption/ionization-time of flight MALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis.

Here we show that major color pattern elements of Heliconius butterfly wings develop independently of wing venation. We recovered a hybrid Heliconius displaying a mutant phenotype with a severe vein deficiency. Although this butterfly lacked most of its wing veins, the large, melanic banding patterns typical of the genus were conserved across the entire wing. The only obvious correlation between vein reduction and pigment patterns was a loss of vein-associated melanin stripes near the distal margin of the wings. We examined the expression of the eyespot-associated transcription factor Distal-less in a banded and a spotted species of Heliconius and found no obvious relationship between protein expression and the band or spot patterns typical of the genus. Together, our results suggest that the melanic bands and spots in Heliconius are unlikely to be derived from an eyespot determination system. We propose that major elements of Heliconius wing pattern formation are based primarily on a complex, whole-wing proximodistal axis system.

Inter-colonial aggression was tested using three colonies of Scaptotrigona bipunctata in a natural setting when their nests were moved and by artificial contact between individuals. Examination of the cuticular lipids of individuals from two colonies kept under identical conditions showed clear differences in their cuticular hydrocarbon profiles. The cuticular lipids were a mixture of hydrocarbons (saturated and unsaturated alkanes and alkenes) within the range of C23-C29. The use of multivariate analysis (PCA and discriminant analysis) showed that seven of the identified surface compounds are enough to separate workers from colonies A and B from each other.

Various cuticular regions from the desert locust, Schistocerca gregaria, were quantitatively analyzed for two cross-linking amino acids, dityrosine and trityrosine, characteristic constituents of the rubberlike cuticular protein, resilin. These amino acids were found in all regions of cuticle investigated, but in widely varying amounts. In fully mature adult locusts the largest amounts of di- and trityrosine were obtained from the prealar arms and wing-hinges, structures possessing long-range elasticity and being involved in energy storage in the flight system. In structures where deformations tend to occur more slowly, such as the clypeo-labral springs and tracheae, di- and trityrosine are less abundant. In sclerotized cuticle from femur and tibia, as well as in cornea and in the highly stretchable intersegmental membranes of mature females, they are only found in trace amounts and are probably unrelated to elasticity. The trityrosine-to-dityrosine ratio in the various cuticular regions vary from nearly equal amounts of the two amino acids to about ten times more dityrosine than trityrosine, indicating that the regions differ in degree of cross-linking; the tracheal wall is the material with the highest trityrosine-to-dityrosine ratio. In some cuticular regions the ratio increases during maturation from newly moulted (teneral) adults to reproductively active locusts; the most pronounced increase was observed for the wing-hinges, and only a small increase was observed for the abdominal tergal plates. In most cuticular regions in fifth instar locust nymphs the contents of di- and trityrosine corresponded to the contents measured for the adult cuticular regions, but only trace amounts of the two amino acids were obtained from the region of the nymphal wing base which corresponds to the wing-hinge containing cuticular region in adult locusts.

In social insects, cuticular hydrocarbons are involved in species, kin, caste and nestmate recognition. Gas chromatography and mass spectrometry were used to compare the cuticular hydrocarbon composition of workers, males and queens of Melipona bicolor. The cuticular hydrocarbon composition of this species was found to consist mainly of C23, C25:1, C25, C27:1, C27, C29:1 and C29, which are already present in imagoes that have not yet abandoned the brood cell. This composition varied quantitatively and qualitatively between and within the castes and sexes. The newly emerged workers and young queens (virgins) had similar cuticular hydrocarbon profiles, which were different from those of the males. When the females start executing their tasks in the colony, the cuticular hydrocarbon profile differences appear. The workers have less variety, while the queens conserve or increase the number of cuticular hydrocarbon compounds. The queens have more abdominal tegumentary glands than the workers, which apparently are the source of the new cuticular compounds.

Morphological, immunocytochemical and ultrastructural methods were used to investigate the role of cells during elastogenesis in the elastic tendon of the chicken wing. Intimate contact of the cell processes with elastic fibers was observed in adult birds. During development there was a sequential appearance of microfibril bundles that became progressively impregnated with amorphous elastin, which eventually predominated in fully developed elastic fibers. The growing elastic fibers were usually enveloped by recesses of the cell surface. The tendon cells were polarized in their association with fibrous components of the extracellular matrix. This arrangement suggests that these cells secrete and organize elastic and collagen fibers to different extracellular compartments. These results show that cells are intimately involved in producing components of different extracellular matrix fibers, in controlling their assembly, and in defining their borders and associations during development.

Biopterin, isoxanthopterin and 6-pterincarboxylic acid were identified in the head of the malaria vector mosquito Anopheles albimanus Weidemann (Diptera: Culicidae) by HPLC. Total pteridine concentrations (TPC) were estimated in heads, body parts (BP: abdomen, legs and wings) and whole bodies of insectary-reared and field-collected females, by spectrofluorometry, to investigate whether they could be used for age determination. Pteridine concentrations diminished with age in both mosquito groups. TPC correlated with chronological age in insectary-reared sugar-fed females (heads: r2 = 0.35, BP: r2 = 0.34, P < 0.001), but lower correlation occurred in blood-fed females (heads: r2 = 0.22, BP: r2 = 0.27). TPC differed among females of the same age fed with blood at different times (P < 0.05), indicating that bloodmeals modify the diminution rate of pteridines with age. Nevertheless, a polynomial significant correlation was documented for TPC and the number of ovipositions (heads: r2 = 0.24, BP: r2 = 0.27, whole body: r2 = 0.52, P < 0.001) in insectary-reared mosquitoes. This correlation was lower in field-collected mosquitoes (heads: r2 = 0.14, BP: r2 = 0.10, P < 0.05), which showed a remarkable pteridine increase in one-parous females. The correlation of TPC in whole body with physiological age was much less (r2 = 0.03). These observations indicate that TPC determination by spectrofluorometry is not a reliable method to estimate the age of An. albimanus females from the feral population.

Capillary gas-liquid chromatography was used to analyse the cuticular hydrocarbons of three triatomine species, Triatoma dimidiata, T. barberi and Dipetalogaster maxima, domestic vectors of Chagas disease in Mexico. Mixtures of saturated hydrocarbons of straight and methyl-branched chains were characteristic of the three species, but quantitatively different. Major methylbranched components mostly corresponded to different saturated isomers of monomethyl, dimethyl and trimethyl branched hydrocarbons ranging from 29 to 39 carbon backbones. Sex-dependent, quantitative differences in certain hydrocarbons were apparent in T. dimidiata.

Effects of short-term UV irradiation were investigated on various cuticular parts of workers and queens of the Oriental hornet, to wit: brown strip, yellow strip and wing. On each preparation of the afore-mentioned, a reading of the relative optical density (ROD) was taken prior to, immediately following, and 15-30 minutes after its irradiation as compared to white light irradiation. The results showed that brief UV irradiation causes changes in the ROD of hornet cuticle, and that these changes in ROD are different in brown than in yellow cuticle. Those in yellow strip are induced by the presence or absence of the active yellow pigment, whose quantity in worker cuticle is different than in queen cuticle, probably due to the various activities in which they are involved during the active season.

Adults, particularly males, of a papilionid butterfly, Papilio machaon hippocrates, emit a fairly strong scent perceivable by humans. We have identified a variety of volatile compounds (hydrocarbons, alcohols, aldehydes, ketones, esters, and so on) from the wings and bodies of both sexes of the butterfly. Male wings secreted n-dodecane, linalool and geranylacetone as major components together with small amounts of camphene, limonene, p-cymene, 2-phenylethanol, n-hexanal, n-decanal, isoamyl acetate, p-allylanisole, 2-pyrrolidone and other characteristic volatiles. The overall profile of volatile compounds detected from male body was quite different from that of the wings. Male body was devoid of camphene, 2-phenylethanol, n-hexanal but instead contained limonene, acetoin, a sesquiterpene hydrocarbon (C15H24, methyl n-octanoate, (E,E)-hepta-2,4-dienal, and another isomer of heptadienal as principal components, of which the last four compounds were specific to the body. All these substances seem to concurrently characterize the male odor. The chemical patterns of compounds found from female wings and body were essentially the same in quality as those of male wings and body, respectively, although their quantities in females were generally smaller than in males. Females, however, had a larger amount of acetamide than males. The chemical compositions of volatiles from the fore and hind wings of males were not greatly different from each other, and every component was considered to be present on all parts of the wings. This suggests that the scent-producing organs or scent-emitting pores are widely distributed on the whole wings. EAG responses of both sexes to 12 selected compounds identified from the butterfly were not strong at a dose of 1 microg, while both sexes showed relatively stronger responses to n-nonanal, methyl n-octanoate, D-limonene and linalool at a higher dose (10 microg). Although sexual difference in EAG response was not prominent, females appeared a little more sensitive, and n-nonanal and acetoin evoked significantly higher responses from females at 1 microg.

The secreted signaling protein Dpp acts as a morphogen to pattern the anterior-posterior axis of the Drosophila wing. Dpp activity is required in all cells of the developing wing imaginal disc, but the ligand gradient that supports this activity has not been characterized. Here we make use of a biologically active form of Dpp tagged with GFP to examine the ligand gradient. Dpp-GFP forms an unstable extracellular gradient that spreads rapidly in the wing disc. The activity gradient visualized by MAD phosphorylation differs in shape from the ligand gradient. The pMAD gradient adjusted to compartment size when this was experimentally altered. These observations suggest that the Dpp activity gradient may be shaped at the level of receptor activation.

As part of an investigation of the functional mechanics of the hind wing of the desert locust Schistocerca gregaria, the Young's modulus of the membrane was measured using a newly developed universal materials test machine capable of testing very small specimens of cuticle, down to 1 mm gauge length. Strain was measured optically. Specimens were cut from various locations around the wing and tested under controlled temperature and humidity. The modulus of the membrane was typically between 1 and 5 GPa, but both this and the membrane thickness varied around the wing, with the remigium and the anal fan showing markedly different properties. The membrane was tested for chitin using two methods: a gas pyrolysis/mass spectrometry assay, and a gold-labelled immunoassay specific to chitin. None was detected, and the membrane may consist of epicuticle alone. The wings were examined for evidence of crystalline material using standard polarising microscopy and an advanced technique that distinguishes between three components of the polarised image. Birefringence was detected in the membrane of the anterior part of the wing, but vanished when the membrane was separated from the surrounding veins, suggesting that it was due to pre-stress rather than to ultrastructure. The implications are discussed.

The elastic tendon of the avian wing has been described by others as a unique structure with elastic properties due to the predominance of elastic fibers in the midsubstance. Further analyses of the tendon have shown it to possess five anatomically distinct regions. Besides the major elastic region, a distally located fibrocartilage and three tendinous regions are present. The tendinous regions connect: (1) the muscle to the elastic region, (2) the elastic region to the fibrocartilage and (3) the latter to the insertion site. The elastic region possesses thick and abundant elastic fibers and very thin, interconnecting collagen fibers. The collagen fibers in the sesamoid fibrocartilage are thick and interwoven, defining spaces occupied by fibrochondrocytes embedded in a non-fibrillar and highly metachromatic matrix. Biochemical analyses have shown that the fibrocartilage has about tenfold the amount of glycosaminoglycans (GAGs) found in the other regions. The main GAG in this region was chondroitin sulfate (CS) (plus keratan sulfate as detected immunocytochemically), while the other regions showed variable amounts of CS, dermatan sulfate (DS) and heparan sulfate. Further analyses have shown that a large CS-bearing proteoglycan is found in the fibrocartilage. The elastic region possesses two main proteoglycans, a large CS-bearing proteoglycan (which reacted with an antibody against keratan sulfate after chondroitinase ABC treatment) and a predominant DS-bearing proteoglycan, which showed immunoreactivity when assayed with an anti-biglycan antibody. The results demonstrate that the elastic tendon is a complex structure with complex regional structural and compositional adaptations, suited to different biomechanical roles.

Chicken hot-drumettes containing 2% cayenne pepper were prepared using three different cooking methods and stored under two different temperatures for "hotness" evaluation. Results demonstrated that the hotness of the samples were highest when products were cooked in a microwave oven, followed by convection oven, and least hot by deep-fat frying. Regardless of the cooking method, the degree of hotness of the hot-drumette decreased drastically during refrigerated storage. Results also indicated that the loss of hotness was associated with the increase of 2-thiobarbituric acid (TBA) values in the product. The addition of antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), rosemary, and sodium nitrate during preparation or vacuum packaging retarded lipid oxidation of chicken hot-drumettes and retarded the loss of hotness of the products during refrigerated storage.

MEF2 is a MADS-box transcription factor required for muscle development in Drosophila. Here, we show that the bHLH transcription factor Twist directly regulates Mef2 expression in adult somatic muscle precursor cells via a 175-bp enhancer located 2245 bp upstream of the transcriptional start site. Within this element, a single evolutionarily conserved E box is essential for enhancer activity. Twist protein can bind to this E box to activate Mef2 transcription, and ectopic expression of twist results in ectopic activation of the wild-type 175-bp enhancer. By use of a temperature-sensitive mutant of twist, we show that activation of Mef2 transcription via this enhancer by Twist is required for normal adult muscle development, and reduction in Twist function results in phenotypes similar to those observed previously in Mef2 mutant adults. The 175-bp enhancer is also active in the embryonic mesoderm, indicating that this enhancer functions at multiple times during development, and its function is dependent on the same conserved E box. In embryos, a reduction in Twist function also strongly reduced Mef2 expression. These findings define a novel transcriptional pathway required for skeletal muscle development and identify Twist as an essential and direct regulator of Mef2 expression in the somatic mesoderm.

Wing cuticle from pharate adult locusts, Locusta migratoria, contains several prominent proteins which occur as minor components or are completely absent in other cuticular regions. Six of the wing-specific proteins have been purified and their amino acid sequences determined by combined use of mass spectrometry and automated Edman degradation. During the sequence determination very long sequence runs (90-121 residues) were necessary in order to establish the primary structure. All the wing-specific cuticular proteins from locusts contain the repeated short sequence motif -Ala-Ala-Pro-Ala/Val-, which is common for all hitherto sequenced cuticular proteins from pharate locusts. Several of the wing-specific proteins also possess an N-terminal region rich in glycine, tyrosine and leucine, characteristic for many locust cuticular proteins. Two of the analysed proteins have a conserved 61-residue sequence in common with a previously sequenced protein from locust wing cuticle and with two proteins from the pharate cuticle of adult Tenebrio molitor. Possible roles for the various sequence motifs are discussed.

Two arsenic compounds, sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4), were tested for their possible genotoxicity in germinal and somatic cells of Drosophila melanogaster. For germinal cells, the sex-linked recessive lethal test (SLRLT) and the sex chromosome loss test (SCLT) were used. In both tests, a brood scheme of 2-3-3 days was employed. Two routes of administration were used for the SLRLT: adult male injection (0.38, 0.77 mM for sodium arsenite; and 0.54, 1.08 mM for sodium arsenate) and larval feeding (0.008, 0.01, 0.02 mM for sodium arsenite; and 0.01, 0.02 mM for sodium arsenate). For the SCLT the compounds were injected into males. Controls were treated with a solution of 5% sucrose which was employed as solvent. The somatic mutation and recombination test (SMART) was run in the w+/w eye assay as well as in the mwh +/+ flr3 wing test, employing the standard and insecticide-resistant strains. In both tests, third instar larvae were treated for 6 hr with sodium arsenite (0.38, 0.77, 1.15 mM), and sodium arsenate (0.54, 1.34, 2.69 mM). In the SLRLT, both compounds were positive, but they were negative in the SCLT. The genotoxicity of both compounds was localized mainly in somatic cells, in agreement with reports on the carcinogenic potential of arsenical compounds. Sodium arsenite was an order of magnitude more toxic and mutagenic than sodium arsenate. This study confirms the reliability of the Drosophila in vivo system to test the genotoxicity of environmental compounds.

We have isolated a cDNA and gene encoding a protein (HCCP66) found in the rigid cuticles of both larvae and pupae of the silkmoth, Hyalophora cecropia. The cDNA encoded a protein similar to cuticle proteins isolated from several other insects and contained a sequence motif similar to one present in a "family" of cuticular proteins from flexible cuticles. The gene had a structure similar to that of cuticle protein genes isolated from Drosophila melanogaster, albeit with a much larger intron that contained three copies of a transposable element-like sequence similar to short interspersed repeated DNA elements (SINEs). A sequence found 5' to the transcription start site matched the Octamer (Oct) cis-acting element. This sequence was capable of binding protein(s) from whole cell extracts of wing epidermis with high affinity and sequence specificity suggesting a role in transcriptional regulation.

The protein encoded by the Drosophila tissue polarity gene, frizzled (fz), is required for both the intercellular transmission and the intracellular transduction of a tissue polarity signal. In order to study the biochemical characteristics of this rare protein, we constructed a hs-fz fusion gene and transferred this to Drosophila tissue culture cells and embryos. Cell fractionation experiments and immunostaining experiments showed that the Fz protein is an integral membrane protein containing an odd number of transmembrane domains, consistent with previous suggestions that it contains seven transmembrane domains. Immunostaining of pupal wings showed that the Fz protein is evenly distributed throughout the wing arguing that the Fz protein does not act as a graded morphogen.

Previously, we isolated an avian protein which we named retinoic acid induced heparin binding factor (RIHB). RIHB is a 121 amino acid secreted polypeptide, rich in basic and cysteine residues (Vigny et al., Eur. J. Biochem. 186: 733-740, 1989). Northern blot analysis indicates that the RIHB gene is transiently expressed during embryogenesis (Urios et al., Biochem. Biophys. Res. Com. 175:617-624, 1991). Here we present an investigation of RIHB expression during early chicken embryogenesis by in situ hybridization and immunofluorescence studies. In the 3-day embryo (stage 20-21), the RIHB transcript is observed throughout the embryo, with the notable exception of the neural tube. At this stage the protein can be visualized in almost all of the basement membranes and around many types of cells. The localization of the RIHB protein does not strictly parallel that of its messenger. Between days 3 and 11 we focused our attention on wing development. The level of both the mRNA and protein decreases during this period but the disappearance is not uniform. The level of both the mRNA and protein decreases during this period but the disappearance is not uniform. The transcript becomes progressively restricted to epithelia and regions surrounding the forming cartilage. In contrast to the transcript, the protein accumulates in the epithelial basement membrane and, interestingly, in the central part of the embryonic cartilage (diaphysis) but not in the distal parts (epiphysis). These data are discussed in relation to the putative role(s) of RIHB in development.

Proteoglycans are ubiquitous extracellular matrix molecules whose role in development remains poorly understood. In the developing chick limb, the nature and possible roles of a number of extracellular matrix proteins is well documented. Much less is known of the biochemical nature, and more importantly, the roles of proteoglycans. Using a panel of monoclonal antibodies (Mabs) which recognise specific epitopes on the constituent chondroitin/dermatan sulphate chains, we show that distinct sub-populations of proteoglycans are dynamically expressed within the limb ectoderm, the ectodermal basement membrane and the limb mesenchyme. In particular, prior to chondrogenesis, chondroitin-6-sulphate-rich proteoglycans containing over-sulphated domains residue predominantly within the mesenchymal extracellular matrix ECM, whilst chondroitin-4-sulphate (C-4-S) is associated with the ectodermal basement membrane and subjacent mesenchymal ECM. At stage 24, C-4-S is also localized in the prechondrogenic condensation. Concomitantly with overt chondrogenesis, the epitopes recognized by the Mabs become restricted to the chondrifying skeletal elements and the undifferentiated distal mesenchyme. The significance of these findings has yet to be elucidated.

An improved method of high resolution two-dimensional gel electrophoresis was used to study the patterns of protein synthesis in the central nervous system (CNS) of late instar larvae of Drosophila melanogaster. A small number of CNSs was radiolabeled with a mixture of 14C-labeled amino acids or with [35S]methionine, and the pattern of labeled proteins was analyzed. One thousand and forty-five polypeptides, 800 acidic (IEF) and 245 basic (NEPHGE), from the CNS of several wild-type strains have so far been separated and cataloged. All these polypeptides were numbered and compared with our catalog of polypeptides from wing imaginal discs, and quantitative or qualitative changes were detected in more than 23% of the polypeptides. When comparing patterns of label of CNSs we found small quantitative differences in the rate of synthesis between individuals of the same strain, not due to sexual differences, and a minute number of quantitative and qualitative differences between groups of individuals of different strains.