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Grid Formation in Hornet Cuticle Induced by Silk Spun during Pupation Jacob S. Ishay* and Shira Kirshboim Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel Received January 12, 2000
Examinations carried out via atomic force microscope (AFM) on the Oriental hornet Vespa orientalis (Hymenoptera, Vespinae) revealed, on the cuticle of the gaster in the region of the yellow stripes, flats bearing a grid in the form of parallel strips. Such configurations, of the same dimensions, were obtained also by superposition imaging of the longitudinal fibrils in the pupal silk weave, which are comprised of a central fibril of fibroin and an outer coating of sericin. In the latter instance, the configurations were revealed in regions where the outer coating of the fibers was disrupted or occurred only intermittently. We conjecture that the silk fibers and flats which encase the 5th-instar larva as it pupates in the dark somehow exert an effect on the morphology of the epicuticle in the metamorphosing pupa and that the effect is achieved via irradiation of heat waves, by an electric charge, or by the induction of a magnetic field. We discuss the possible implications of the presence of grids on the cuticles of adult hornets and also their possible connection with solar cells and light polarization. Introduction From a systematic standpoint, the social wasps and hornets (Vespinae) belong to the very large group of insects (Holometabola) that undergo a complete metamorphosis; that is, insects that have larval and pupal stages between the egg and adult stages. In our case, the mature larval stage (5th instar) goes into diapause (pupal stage) after enwrapping itself in a silk weave comprised of fibers and interspersed flats. The spun silk originates from a labial gland which is present both in the pupating larva as well as in the adult hornets (workers and young queens but not the drones), the latter using this secretion to bind together particles of matter, like pieces of wood or paper, used to build the cell walls and other parts of the comb.1-3 The comb building itself is intended to prepare sites for oviposition and subsequent development of the brood. To this end, cells are built upsidedown, with their outlet facing downward, in the direction of the gravitational force;4,5 in these “inverted” cells, the queen lays eggs that give rise, depending on the season, to worker, drone, and queen larvae6 that undergo several larval stages called instars. The mature larva (5th instar), prior to pupate, encases itself in a silk weave, positioning itself in its home cell so that all or most of its body is inside the cell. The walls of the cells housing the brood have slits that enable light to pass between the cells.7 The pupating larva first spins a silk cap which, inter alia, seals the cell entrance and thus ensures that the larva will not fall out of the cells if it loosens its hold on the cell wall. Next the pupating larva spins the rest of the silk weave, namely, a “sleeve” around its body. The spinning of a silk cap is understandable for the * Corresponding author. Telephone: +972-3-6409138. Fax: +972-36409113. E-mail:
[email protected].
aforementioned reason, but why spin a “sleeve” around the body? Well, the spinning of a silk weave around the entire body is understandable in Lepidoptera which pupate in isolation and also in Hymenoptera that pupate discretely, such as ants, but does seem redundant in insects such as hornets which pupate in comb cells and maintain a steady nest temperature, unless the vespan silk serves additional purposes. In this regard, we now know that vespan silk possesses the properties of an organic semiconductor.8 Furthermore, upon an increase in temperature and in the dark the silk produces an electric current,9 which means that it is endowed with thermoelectric (Seebeck) properties. We have additionally found the vespan silk to be photovoltaic11 and capable of very high electric capacitance.12 Larvae of the Hymenopteran-Aculeata produce silk in their labial glands.13 In Vespinae (and other insect orders) the posterior gland region secretes a group of proteins commonly known as fibroin as well as some small proteins which together form the core of the silk fiber. This core is enveloped by a mixture of coat proteins of the sericin type. The major component (by weight) of the silk thread is fibroin. Its amino acid composition differs greatly in various insect species. Some fibroins, e.g., those in the silkworm Bombyx mori (Lepidoptera) have a molecular weight of about 360 000 while in the larva of this species the dominant sericins have a molecular weight range of 80 000-300 000.14 In the silkworm, the amino acid composition of fibroin was obtained by Tashiro et al.15 and Prudhome et al.16 Sericin is heterogenic. Histochemical analysis revealed the presence of some four distinct sericin layers around the fibroin core.17,18 The amino acid composition of sericin polypeptides has been partially purified.14,19 Structurally, the silk fiber is composed
10.1021/bm000290b CCC: $19.00 © 2000 American Chemical Society Published on Web 04/09/2000
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of a core of fibroin arranged as fibrils “running” lengthwise and a coat of sericin arranged in transverse striations as fibrils “running” crosswise, with the ratio of the former to the latter being 4:1.20 Apart from the silk fibers, the weave also contains plates or flats which interconnect between the fibers, and it seems likely that the interior of the cell housing the pupating larva is devoid of light. Apparently, the pupating larva wraps itself in a silk weave in order to create a “clean room” beneath the weave so as to enable the production of the new cuticle of the future imago, and this occurs without the intrusion of air currents or dust particles which could hamper the creation of the cuticle.21 Indeed, in earlier trials intended to cause the larvae to pupate without a silk weave, we removed the young pupae from their silk weave and placed them in hollows of suitable dimensions bored in plastic or in wood. However, these trials failed completely, the transplanted pupae all dying within a day or two,22 albeit subjected to the necessary darkness and optimal temperature.23 In the present study, we investigated the morphology of the cuticle of adult hornets, attempting to ascertain the nature and origin of the various surface configurations observed. Materials and Methods Combs of the Oriental hornet were collected from the natural habitats both in the Tel-Aviv metropolitan area as well as in the Ein-Gedi region and this by a method previously described.24 Processing for electron microscopic observations was undertaken on strips of silk caps removed from puparia in the natural nest combs and on strips of yellow cuticle removed from the gaster of hornets after killing them by freezing. Samples to be studied by electron microscopy were rinsed briefly in 0.1 M cacodylate buffer solution and then fixed in a mixture of glutaraldehyde 2% (GA) and 2% acrolein in cacodylate buffer for 24 h. For field emission electron microscopy (FE-SEM) the specimens were next prepared according to the tannic acid/arginine/osmium tetraoxide noncoating technique.25,26 Dehydration with ethanol was followed by critical point drying (CPD) in liquid CO2. SEM observations were carried out with JEOL FE-SEM, type 6301F, operated at 2-3 kV. Additionally, some specimens were coated with a layer of gold of approximately 30-40 nm in thickness by a Polaron SEM coating unit E 5100 and finally examined in a JEOL SEM-35 at 25 kV. Small portions of previously studied FE-SEM samples, prepared according to the glutaraldehyde/tannic acid/arginine/osmium tetraoxide noncoating technique, were carefully oriented for embedding in Epon. Ultrathin sections were post-stained with uranyl acetate/lead citrate and observed in a Philips transmission electron microscope (TEM), type CM 100, operated at 60 kV. The atomic force microscope (AFM) method was chosen as a complementary method to SEM in respect to micro- and nanoscale measurements on the hornet material.27,28 The microscope for the AFM procedure was a model Thermomicroscope (Park Scientific Instruments and Topometrix). The advantage of examination with this instrument is, inter alia, the lack of need to coat the specimen to be photographed, and thus the specimen is fastened upon the arm of the microscope and adjusted for direct observation.
Results A strip from a silk cap photographed via SEM is shown in Figure 1A. One can see silk fibers about 10 µm in diameter
Figure 1. (A) SEM picture of silk fibers and flats. The fibers are usually intact but in some short sections have transverse annuli suggesting that the coat is interrupted by ringlike gaps. (B) Single fibers with annuli. For more details see text in Results. (C) Some of the annuli are incomplete. The gaps are not uniform. For details see text.
and also the flats interconnecting some of the fibers and filling the gaps between fibers. The visible fibers are generally intact and smooth, but in some sections, we can clearly see transverse ribbons suggesting that the coat (made of sericin) is interrupted by ringlike gaps (see points 1, 2, and 3). These are the ring gaps mentioned in the figure. Similar annuli can be discerned intermittently on exposed parts of the inner fibrils, mainly on inner segments of the silk enwrapping the pupating larva. Single fibers with exposed inner part are shown in Figure 1B,C. The shown fibers were picked at random. The rings (and therefore the
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Figure 2. Typical temperature dependence of the electrical resistance of the hornet cocoon cap plotted as ln R vs 1000/T (in K). As can be seen, there is a sharp (approximately 3 orders of magnitude) drop in resistance with temperature increase from 1 to about 21 °C, following which there is a plateau (between 21 and 32 °C) that coincides with the optimal biological temperature and hornet activity. Upon further increase of the temperature from 32 °C and up to 50 °C, there is a sharp increase in the resistance. As a control, we measured the resistance at the same temperature range (from 1 to 50 °C) of two resistors (of 1.19 × 107 and of 1.20 × 109 Ω).
gaps) on the outer coating of the fiber are not of uniform width even on the same fiber, are not equidistant, nor are they of the same number on each fiber. Moreover, some of the annuli are incomplete (see Figure 1C). Neither are the fibers themselves, in the ribboned sections, of uniform diameter, but rather vary between 7 µm (part B) and 5 µm (part A) in diameter. Note that at magnifications up to ×2500, one cannot see the fibers in any greater detail. Another view of such “disrupted” fibers, again via SEM, is shown in Figure 3A. The majority of fibers are smooth and unmarked but one is ribboned (arrow at center of picture; magnification ×1000). In part B, at ×5000 magnification, one can see the same site marked by an arrow. Now we can see the inner fibrils of fibroin which were revealed beneath the disrupted regions in the outer coat of sericin. In part C, it is seen once more at higher magnification (×10 000). Sections prepared for and viewed by TEM are given in Figure 4. In part A, we see a cross section through a single silk fiber with “faults”. The cross section here is through the core of the fiber, as indicated by label C, and in its center one can see a sparse region whose borders seem to be electron dense (as indicated by a black granulation and marked by arrows). Along the fiber one can see intermittently dark and light regions extending longitudinally. Outside the core (right of picture) we see the coat (label D) with an interruption (the “faulty” region) which is marked as label E and is packed with filling material which also infiltrates
Figure 3. (A) At the center of a fiber with disrupted coat. For more details see text. (B) By SEM, the inner fibrils of fibroin which were revealed beneath the disrupted regions in the outer coat of sericin. (C) By SEM, the fibrils at ×10 000 magnification running along the fiber of fibroin. For details see text.
underneath the disrupted edges of the coat. Around the external surface of the coat there is a dark region which stains as electron dense. In part B, we see a section similar to that in part A, but at higher magnification. Here one can see the inner fibroin fibrils extending longitudinally and also the dark region around the sericin coat, which extends both along the border between the sericin and the fibroin, as well as on the outside of the sericin coat. Two more cross sections of the core are shown in Figure 5. The upper cross-section is through the core of the silk fiber, while the bottom one is through a more external region. In both sections, one can see the fibrils running longitudinally
Grid Formation in Hornet Cuticle
Figure 4. Cross section viewed by TEM. For details see text.
at slight distance apart. The width of the fibrils is about 150 nm, and although they proceed longitudinally, they display also transverse, intermittently light and dark regions, as seen mainly in part B. In recap, Figures 1 and 3-5 offer pictures of the silk fibers taken through SEM and TEM. In Figure 6, we provide pictures taken via AFM of the cuticle of a worker hornet. In part A, the viewed field is reminiscent of a grid (supposedly a light polarizer in the range of the UV-B light), that is, we see a smooth surface criss-crossed with longitudinal and transverse stripes. The grid is about 4 µm wide. Between each two such grids there is a prominent region bearing furrows of various heights. Part B provides a magnified view of the grid surface. We can now see quasi-fibrils extending longitudinally which are interrupted by transverse stripes. The same, at higher magnification, is given in part C. Now we can see a single, albeit actually double fibril whose width is about 0.3 µm. The fibrils in this and the previous picture appear to have a metameric structure (i.e., an arrangement into the metameres by a serial repetition of the structural pattern), which is fairly uniform, and they extend longitudinally at some remove from one another. Discussion The exact chemical composition of the silk is not known yet. Preliminary analysis of the hornet silk was accomplished using Fourier transform infrared (FTIR) spectroscopy according to the method described earlier.29
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Figure 5. Cross section of the core seen by TEM. For details see text. Fiber orientation along the lines.
Silk caps were cut and removed from the cell or hornet pupae in their combs and small square pieces (3 × 3 mm) were analyzed in an Equinox spectrometer (Bruker Ltd., Ettlingen, Germany) equipped with IRscope II with MCT (mercury calcium telluride) spectral range 120000-600 cm-1 and a special objective lens for IR (magnification ×15). Data acquisition and manipulation were performed on a PC using Opus/IR software (Bruker Ltd.). The dry silk samples were placed on a CaF2 crystal and the absorbance was recorded in the range 4000-1000 cm-1. Wide zones of silk caps (containing both fiber and plates) were scanned, and by selection of a narrow aperture (0.2), silk fibers were scanned individually. Some preliminary results regarding the FTIR spectroscopic analysis of hornet silk caps have already been published.30 Thus, FTIR analysis of intact silk caps indicated absorbing peaks of carbonyl groups in the region 1820-1660 cm-1, amides in the region 3500 cm-1, C-H bonds in linear (3000-2850 cm-1) and in aromatic groups (3150-3050 cm-1), and CdC bonds of aromatic groups (1600-1400 cm-1). Several peaks are flattened relative to the general profile of the silk cap in the ranges 3200-3900 or 23001750 cm-1. In contrast, fibers showed a “noisy” spectrum, probably due to thickness of the fibers relative to the absorbance in the range 3700-2800, 2400-2300, 23001750, 1750-1700, and 1500-1000 cm-1. No characteristic structure can be drawn from the data yet. However, fibers and plates appear to differ in the structure probably due to different content (or blending) of the protein(s) involved.
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Figure 6. Grid seen in the adult cuticle. For details see text.
Preliminary unpublished data concerning the dissolution of the silk caps in hot alkaline saline indicated that protein(s) constitutes the major building material. Furthermore, silk plates and the external layer of the fibers dissolved more readily than the core of the fibers. These data along with the IR spectroscopic data suggest that the silk caps are not homogeneous in their protein composition; that is, more than one protein is involved, and their distribution among the various structural components of the silk caps is not uniform. The present paper describes structures produced by the pupating larva of the Oriental hornet, namely, silk fibers and
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silk flats. The manner in which these structures are deposited in the silk weave spun by the pupating larva appears to be random or fortuitous but actually is properly designed. In fact, structures of the same size and shape make their appearances also in the cuticle of the imago that has developed within the silk weave. The silk of the hornet is a thermoelectric material which produces, in the dark, an electric current amounting to several scores of nanoamperes (nA). This production is cyclical: that is, with a rise in temperature, the current increases up to a certain maximum (32.3 °C) beyond which the current diminishes, and upon cooling the current drops while the resistance increases (see Figure 2).30 This current discharges upon the electrode which is connected to the studied specimen by conductive silver, which renders it a Schottky connection, that is, a connection between an organic semiconducting material and a metal. How the current discharge transpires in nature is still unclear, but we suppose that it does take place in the natural nest as well, although, perhaps, at a different pace. The silk fiber is composed of an inner core, arranged as an uninterrupted fibril, and an outer coat which is interrupted at close intervals. Since the outer coat enwraps the inner fibril crosswise, the interruptions in the coat frequently assume the shape of a ring or annulus (Figure 1). These interruptions expose to view the micromorphology of the fibrils comprising the coat which superficially appear as metameric fibrils running lengthwise (Figure 3B,C) and measuring about 150 nm in diameter. In cross sections of the silk fibers (Figures 5 and 6), appearance of the fibrils is also as afore-described, but in such sections one can, of course, observe aggregates of fibers running lengthwise and can attribute to them the structure of a unit. Something that has not yet been done, but should prove worthwhile, is to focus on the outer covering of the silk fibersthe coatswhich allegedly is composed of the protein sericin. In the two figures appearing in Figure 4, we can see that the coat is fairly thick (occupying about a third of the diameter of the fiber), contains an amorphous material, and is itself encased in an electron dense sheath. It stands to reason that the sericin coat is the electric capacitor of the silk fiber, which insulates it from the surroundings. To the extent that there is any leakage, this takes place, in our opinion, in the disruption points in the coat, which enable the core filaments to come in direct contact with the “flats” of the coat (Figure 1A, Figure 4A). What is transmitted through the gaps in the coat? We conjecture that what the immediate environment of the developing pupa requires and acquires is heat, inasmuch as the developing creature lacks muscles or significant metabolic energy while it is undergoing metamorphosis and up to its maturation into an imago; neither does it feed throughout this period. The silk cap temperature is not higher than the ambient temperature, so it is rather unlikely that it accumulates heat or chemical substances that release heat.10 What the silk cap does accumulate and release is electric energy, and it is this which flows within the silk fibers and this is what the fibers release in vitro, thus increasing the temperature. As long as the fibers are encased in a coat that
Grid Formation in Hornet Cuticle
Figure 7. Silk caps measured thermographically showing diverse temperatures while the pupae extract heat from the caps according to their needs. For more details see text. The numbers represent the temperature on the silk cap surface.
insulates them from their surroundings, the electric current can flow through them without appreciable loss. However, when there are gaps or breaches in the insulating coat, as shown in Figures 1, 3, and 4, the possibility for a leak exists, provided something is there to absorb the leaking current. This something, in our case, is the amorphous material inside the fiber, whose high resistance converts the electric current to heat (much the same as an electric oven, with its thin filaments of high electric resistance, converts electricity to heat). So far we have pinpointed one clear role of the silk weave, namely, to retain optimal temperature for the pupa during its development and replace any heat loss. Evidence that vespan silk produces and conducts electric current was already presented in earlier publications.31-34 Furthermore, we now have evidence that the pupa draws heat from the silk cap, as witness the thermographic picture which clearly shows that the center of the cap on the outside has a lower temperature than the ambient temperature, while the pupa inside the silk weave maintains a uniform temperature (Figure 7).10 In a picture taken via AFM we find, on the cuticle of the adult hornet, as part of the epicuticle, formations similar to those along the cores of the silk fibers of the pupating larva (Figure 6). In all three pictures of Figure 6, we see fibers of the same size and shape as those “peeking” through the breaks in the coats in Figure 3. Possibly, the cuticle of the adult produces, independently, formations similar to those on the filaments of the fibroin core. We, however, propose another explanation, namely, that the silk fibers encasing the pupa during the critical phase of metamorphosis, in which the cuticle of the future imago is laid down, induce the epicuticle to create formations similar to those in the inducing agent, that is, formations in the imago duplicating those on the fibroin filaments. We have yet to ascertain whether the induction in this case is via an electric charge, heat waves, a magnetic field, or some other mechanism. Remember that the formations under discussion resemble a grid, and one may ponder the need for the adult to have such formations on its epicuticle. In this regard, we point out that the overall shape of the grid network, in which the longitudinal lines
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are numerous and close to one another, while the differing density of the matrix material produces quasi-latitudinal lines, creates a system that can only absorb polarized light.35,36 Insects are able to navigate in polarized light, and this has been extensively investigated in the honeybee.37-41 It has long been known also that hornets perform digging activities in the nest mainly during the noon hours, when the solar irradiation is at a maximum, including that of UV.30 Also long known is the fact that winged insects navigate their flights, even on cloudy days, in reliance on polarized light. Yet ours is a first-ever report of gridlike formations on the cuticle of an insect, and we are confident that in future there will be additional reports on cuticular flats that polarize sunlight and on organs geared to absorb light, like the vespan extraretinal photoreceptors.42 Hopefully, the activities of such surfaces and organs will prove to be coordinated. It was earlier published43 that exposing dead hornets (at room temperature) to light pulses from a halogen lamp triggers the appearance of a voltage on the order of 10-100 mV between the electrodes. This voltage is linearly dependent on the intensity of the incident light, at least for the relatively low intensities (few mW/cm2), with blue light, around 450 nm, being the most efficient. This is clearly a photovoltaic effect. It is worth mentioning that at noon, when the hornet activity reached maximum, this coincides with the maximal UV and blue solar radiation. The discharge curve of the hornet cuticle conforms to that of a capacitor in that it has the shape of an exponential curve, according to the formula I(t) ) I0e-t/τ where I0 is the initial current. This shape of the curve indicates not only that the hornet possesses electric energy but also that this electric energy is utilizable just as much as the energy stored in a capacitor or a battery. Admittedly the curve was obtained at optimal temperature, but even at other temperatures a similar curve is obtained, albeit with differential initial values. The capacitance computed for a single hornet is quite substantial, amounting to millifarads (mFs). Indeed capacitance values of this order of magnitude and at such volume (≈0.429 cm3), i.e., including the cuticle and all the organs within it, conform either to “good” dense capacitors or to batteries. This leads us to believe that the hornet possibly stores electrical energy by means of an electric field (as a capacitor does) or, alternatively, produces it from a chemical reaction (as a battery does). Inasmuch as we can repeatedly recharge the hornet after several discharges (by keeping it overnight or for a long time in low temperature and darkness), it is reasonable to conclude that this vespine response is reversible, so that in this respect it can be regarded as a “battery”. In the natural habitat, hornets tend to leave their nest many times daily during their life. In the course of these sorties out of the nest the hornets pass from conditions of darkness with fixed temperature and humidity to conditions of direct insolation with variable temperature and humidity. It is important to mention that changes in illumination were previously bound to influence the capacitance.43
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Table 1. Capacitance Values Obtained by Two Different Methods temp (°C) 15 15 15 15 15 15 25 25 25 25 25 25 35 35 35 35 35 35
measurement no.a 1A 1B 1C 1D 1E 1F 2A 2B 2C 2D 2E 2F 3A 3B 3C 3D 3E 3F
τ (s)
R (Ω)
C (mF)b
C (mF)c
960< 540 240 300 540 240 360 120 420 180 180 300 180 360 300 120 240 360
2.84 × 5.30 ×104 3.11 × 104 3.96 × 103 1.00 × 105 8.43 × 103 1.04 × 104 2.45 × 103 2.64 × 104 3.48 × 103 4.55 × 104 5.02 × 103 6.52 × 104 5.81 × 103 3.11 × 105 3.16 × 103 2.14 × 105 2.57 × 104
34.00 101.00 7.70 75.00 5.40 28.00 34.00 49.00 16.00 52.00 3.90 60.00 2.70 62.00 1.00 38.00 1.10 14.10
20.17 54.00 7.54 67.00 1.40 18.44 25.23 84.05 13.40 61.42 3.14 59.41 35.2 49.3 1.06 76.19 3.07 11.79
104
a Key: A, 1 silk cap, queen; B, 5 silk caps, queens; C, 1 silk cap, drone; D, 5 silk caps, drones; E, 1 silk cap, worker; F, 5 silk caps, workers. b First method of calculation by resistance. c Second method of calculation by voltage.
Measurements confirm that a hornet is indeed capable of utilizing its capacitance to store an electric charge, that is to say, to store energy. It is reasonable to assume that this energy has its application in vespine life. The capacitance measured in the silk caps is higher by at least 1 order of magnitude than that measured in the cuticle (see Table 1).12 Our findings have yielded very high capacitance values and these results received dual confirmation from two different methods of computation, each based on the measurement of independent size factors. Such high values of capacitance are obtainable normally only via commercial electrolytic capacitors having a mass that is greater, by several orders of magnitude, than that of the silk caps. We should note that a hornet silk cap (the convex area measured) weighing on the average about 5 mg, has a surface area of about 120 mm2 (queen pupa), 110 mm2 (male pupa) and 100 mm2 (worker pupa)44 and a thickness of about 40 µm.45 The short circuit currents which we were able to produce in the course of the measurements suggest that it may be possible to charge and discharge the silk caps with electric energy. This brings us to the question of why the difference between the cuticle and the silk is so great in this respect? It appears to us that the adult hornets are charging their cuticle while exposed to solar radiation and are later discharging part of the acquired electrical energy on the silk caps which thus act as a reservoir or a sink for electrical energy for the benefit of the entire colony. References and Notes (1) Imms, A D. Entomology; Methuen: London, 1960. (2) Spradbery, J. P. Wasps; Sidgwick & Jackson: London, 1973. (3) Edwards, R. Social wasps; The Rentokil Library, Rentokil Ltd.: East Grinstead, England, 1980. (4) Ishay, J.; Sadeh, D. Direction finding of hornets under gravitational and centrifugal forces. Science 1975, 190 (4216), 802-804. (5) Ishay, J.; Sadeh, D. Geotropism of hornet comb construction under persistent acceleration. BehaV. Ecol. Sociobiol. 1977, 2, 119-129.
(6) Matsuura, M.; Yamane, S. Biology of the Vespine wasps; SpringerVerlag: Berlin, 1990. (7) Darchen, R. Biologie de Vespa orientalis. Les premiers stades de de´veloppement. Insectes Soc. 1964, 11, 141-157. (8) Gutmann, F.; Keyzer, H.; Lyons, L. E.; Somoano, R. B. Organic semiconductors. Part B; R. E. Krieger Pub. Co.: Malabar, FL, 1983. (9) Litinetsky, L.; Steinovitz, H.; Ishay, J. Thermoelectric properties of hornet silk caps during different stages of pupation. Physiol. Chem. Phys. Med. NMR 1998, 30 (1), 129-140. (10) Litinetsky, L.; et al. Manuscript in preparation. (11) Ishay, J. S.; Benshalom-Shimony, T.; Ben-Shalom, A.; Kristianpoller, N. Photovoltaic effects in the Oriental hornet. J. Insect Physiol. 1992, 38 (1), 37-48. (12) Ishay, J. S.; Barenholz-Paniry, V.; Chernobrov, H. L. The electrical capacitance in hornet silk cocoon cap. Physiol. Chem. Phys. Med. NMR 1994, 26 (4), 329-342. (13) Sehnal, F.; Akai, H. Insect silk gland types, development and function, and effects of a mental factors and morphogenetic hormones on them. J. Insect Morph. Embryol. 1990, 19, 79-132. (14) Gamo, T.; Inokuchi, T.; Laufer, H. Polypeptides of fibroin sericin secreted from the different sections of the silk gland in Bombyx mori. Insect Biochem. 1977, 7, 285-295. (15) Tashiro, Y.; Ohtsuki, E.; Shimatzu, T. Sedimentation analysis of native silk fibroin in urea and guanidine-HCl. Biochim. Biophys. Acta 1972, 257, 198-209. (16) Prudhome, J. C.; Couble, P.; Garel, J. P.; Daillie, J. Silk synthesis. Compr. Insect Physiol. Biochem. Pharmacol. 1985, 17, 571-625. (17) Komatsu, K. Studies on dissolution behaviors and structural characteristics of silk sericin. Bull. Sericult. Exp. Stn. 1975, 26, 135256. (18) Komatsu, K. Silk. II. Sericin chemical structure. Sericologia 1981, 21, 307-332. (19) Sinohara, H. Glycopeptides isolated from sericin in the silkworm, Bombyx mori. Comp. Biochem. Biophys. 1979, 63B, 87-91. (20) Ochiai, S. Comparative studies on embryology of the bees - Apis, Polistes, Vespula and Vespa, with special reference to the development of the silk gland. Bull. Fac. Agric. Tamagawa UniV. 1960, 1, 13-45. (21) Shabtai, Y.; Ishay, J. Hornet silk caps maintain a clean room environment: A device for filtering out bacteria and dust particles. Comp. Biochem. Physiol. A. 1998, 120, 565-570. (22) Ishay, J. Unpublished observation. (23) Ishay, J.; Ruttner, F. Die thermoregulation im Hornissennest. Z. Physiol. 1971, 72, 423-434. (24) Ishay, J. Observations sur la biologie de la Gueˆpe orientale Vespa orientalis en Israe¨l Insectes Soc. 1964, XI (3), 193-206. (25) Jongebloed, W. L.; Kalicharan, D. Low voltage visualization of glycocalyx(-like) structures in various biological tissues. Beitr. Elektronenmikr. Direktabb. Oberfl. 1996, 29, 201-212. (26) Kalicharan, D.; Jongebloed, W. L.; Los, L. J.; Worst, J. G. F. Application of tannic acid non coating technique in eye research: Lens capsule and lens cataractous lens fibers. Beitr. Elektronenmikr. Direktabb. Oberfl. 1992, 25, 201-205. (27) Barkay, Z.; Krupke, R.; Shechter, R.; Deutscher, G.; Peled, E. AFM as a complementary method to SEM for micro and macro-structure measurements. Second Agil conference on material science and technology. Ramat-Gan, Israel, Nov 1998. (28) Joachim, C.; Bergoud, C.; Pinna, H.; Tang, H.; Gimzewski, J. K. Is there a minimum size and a maximum speed for a nanoscale amplifier? In Aviram, A., Ratner, M., Eds.; Molecular Electronics. Ann. New York Acad. Sci. 1998, 852, 243-256. (29) Smith, B. C. Fundamentals of Fourier Transform Infrared Spectroscopy; CRC Press: Boca Raton, FL, 1996. (30) Ishay, J. S.; Litinetsky, L.; Rosenzweig, E.; Kirshboim, S.; BenShalom, A.; Steinberg, D.; Shabtai, Y. The hornet - one of nature’s solar cells: the thermophotovoltaic (TPV) properties of its cuticle and silk. The Sede Boqer Symposium on Solar Electricity Production, Ben Gurion National Solar Energy Center. DEEP 1999, 99/38, 1349. (31) Benshalom-Shimony, T.; Ishay, J. S. Electrical capacitance and current of the Oriental hornet silk cocoon cap. Comp. Biochem. Physiol. 1990, 97, 555-563. (32) Ishay, J. S.; Benshalom, A. Electrical properties of hornet silk: the influence of humidity and ether. Physiol. Chem. Phys. Med. NMR 1992, 24 (4), 323-328. (33) Ishay, J. S.; Barenholz-Paniry, V. Thermoelectric effect in hornet silk and thermorgulation in hornet’s nest. J. Insect Physiol. 1995, 41, 753-759.
Grid Formation in Hornet Cuticle (34) Ishay, J. S.; Barenholz-Paniry, V.; Bitler, A. Electric energy transfer in hornet silk caps: A quantitative approach to the dynamics of charge release. Physiol. Chem. Phys. Med. NMR 1997, 29 (2), 231-241. (35) Robinson, L. C. Physical Principles of Far-Infrared Radiation; Academic Press: New York, 1973. (36) Brosseau, C. Fundamentals of Polarized Light. John Wiley & Sons: New York, 1998. (37) Lindauer, M. Sonnenorientierung der Bienen under der A ¨ quatorsonne und zur Nachtzeit. Naturwissenschaften 1957, 44 (1), 1-6. (38) Goldsmith, T. H. The visual system of the honeybee. Proc. Natl. Acad. Sci. U.S.A. 1958. 44 (2), 123-126. (39) Goldsmith, T. H.; Fernandez, H. R. Some photochemical and physiological aspects of visual excitation in compound eyes. In The functional organization of the compound eye; Berhard, C. G., Ed.; Pergamon Press: Oxford, England, 1966; pp 125-143.
Biomacromolecules, Vol. 1, No. 2, 2000 231 (40) von Frisch, K. The dance language and orientation of bees. Belknap Press of Harvard University Press: Cambridge, MA, 1967. (41) Wilson, E. O. The Insect Societies; Belknap: Harvard, MA, 1971. (42) Goldstein, O.; Ishay, J. S. Morphology of a putative new peripheral photoreceptor in social wasps. Physiol. Chem. Phys. Med. NMR 1996, 28 (4), 255-266. (43) Shimony (Benshalom) T.; Ishay, J. Electrical capacitance in hornet integument: Frequency, light and temperature dependence; possible p-n junction effects. Physiol. Chem. Phys. Med. NMR 1984, 16, 333-349. (44) Ishay, J. Unpublished measurements. (45) Ishay, J.; Ganor, X. Unpublished results, 1990.
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