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Hornet silk: thermophysical properties
Journal of Thermal Biology 27 (2002) 7–15
Hornet silk: thermophysical properties J.S. Ishaya,*, L. Litinetskya, V. Pertsis1, D. Linskyb, V. Lusternikb, A. Voronelb a
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 Ramat-Aviv, Israel Department of Physics and Astronomy, Sackler Faculty of Exact Sciences, Tel-Aviv University, 69978 Ramat-Aviv, Israel
b
Received 2 September 2000; accepted 3 February 2001
Abstract Thermoelectric measurements performed on strips of hornet silk have shown marked rise in the electric charge upon increase of temperature between 208C and 338C. The increase was dependent on the relative humidity (RH), occurring only at RH above 90%. In tests of heat capacity, hornet silk was found to possess a specific heat of over 2 J/g K within a temperature range of 5–408C. Observation carried out via transmission electron microscopy (TEM) on silk sections reveal that the silk bears lengthwise electron-dense (dark) stripes separated by light-colored stripes. We assume that in the warm daytime hours, hornet silk can longitudinally transport water that serves to raise the RH and to provide the vaporization needed for nest thermoregulation, whereas at night the very same silk provides an electric charge and longitudinally transfers current and heat. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Hornet silk; Specific heat; Thermoregulation; Calorimetry; Phase transition
1. Introduction The space comprising the nest of the Oriental hornet, Vespa orientalis (Hymenoptera, Vespinae), houses both the adult hornet population as well as combs bearing the brood. The comb brood cells contain larvae, which, upon maturation, commence pupating by weaving a silk cocoon around their body. At first, each such pre-pupal stage spins a silk cap that seals its comb cell outlet from below (the cells having been built upside-down). At this stage, the cell outlet is the only portal into the cell that has been left unbuilt, whereas all the walls as well as the roof (actually bottom) of the hexagonal comb cell have already been built ab initio (by the workers). After completing the silk cap, the pupating larva continues weaving a silk sleeve around itself so that ultimately it becomes entirely encased in a silk weave, comprised of two separable layers (Duncan, 1939). At closer inspec*Corresponding author. Tel.: +972-3-640-9138; fax: +9723-640-9113. E-mail address: [email protected] (J.S. Ishay). 1 Part of a Ph.D. Thesis to be submitted to the senate of Tel Aviv University.
tion, the silk weave is shown to be composed of a ‘stringy’ element}actually one elongated winding silk strand}and additionally also dense, plaque-like flats (Litinetsky et al., 1998; Ishay and Kirshboim, 2000a; Kirshboim and Ishay, 2000). The strings comprising the stringy element are each composed of a central fibril}the core}made up of fibroin proteins (Rudall and Kenchington, 1971; Brunet and Coles, 1974) and, a surrounding coat made up of sericin proteins that are arranged in transverse, with dispersed apparently disconnected annuli (Ishay and Kirshboim, 2000b). Once we have elucidated the manner in which thermoregulation is achieved in the hornet nest, we deemed it highly interesting to compare it to the specific heat of various inorganic and organic compounds including those in various living organisms, whose bodies are, in fact, made up of tissues comprised of organic matter. The literature boasts lately of numerous data on this subject. Inter alia Sabbah and Ider (1999) investigated, by various techniques the heat capacity of small organic molecular compounds (carboxypyridinic acid); Graziano et al. (1998) studied the heat capacity of globular proteins; Miyahara et al. (1999) determined the specific heat of chicken meat of variable fat content; and
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Kobashi and Oguni (1997) investigated the phasetransition states in a chlorocyclohexane–bromocyclohexane system. The above investigations focused on materials, mostly on organic ones. In contrast, Harak et al. (1996) addressed the issues of the metabolic cost of entire organisms in terms of the number of calories spent by an insect when activating its wings or part of the body}in this case the insects Tenebrio molitor and Galleria mellonella; Fahrenholz et al. (1992) concentrated on calorimetric issues in various castes of the honeybee; and Schmolz et al. (1999) found interest in calorimetric measurements in flying hornets. As for ourselves, we have been investigating thermoregulation in nests of the Oriental hornet since 1967 (Ishay et al., 1967) and undertook to ascertain the role, which the silk produced by hornet larvae plays in this process. By now, we know that this silk is made of proteins which make up its fibrilar moiety, namely, fibroin proteins comprising the core and sericin proteins comprising the coat of each fibril. The silk of hornets has been shown to possess electric properties. Thus, upon exposure to light (blue or UV), the silk produces a voltage of several scores to several hundreds of millivolts (mV) (Ishay et al., 1992), while in the dark, the silk produces a current of several scores of nano amperes (nA). Both the current and voltage levels are dependent on the relative humidity (RH) and are optimal when the latter is maximal, that is, close to 100% (Sverdlov et al., 2000). The present study investigated the dependencies between the specific heat of the silk and its thermoelectric properties.
2. Materials and methods Pre-pupal silk caps were obtained from hornet brood combs collected from natural nests in the field. The silk was gathered as previously described (Ishay, 1975) and stored under refrigeration at 48C till used for the following purposes: (1) for observations via light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM), as previously described (Jongebloed et al., 1999); (2) for measuring the thermoelectric current, as previously described (Litinetsky et al., 1998); and (3) for thermophysical study geared to ascertain the specific heat of the silk caps. A large number of silk caps having a total weight of 0.524 g were packed into a preliminarily calibrated container. Specific heat of the silk sample was measured in the range of temperatures from 268C up to +808C. The most accurate values of specific heat and enthalpy of phase transition have been determined by adiabatic calorimeters (Voronel, 1974; Voronel et al., 1988; Chekhova et al., 2000). To this end, an adiabatic calorimeter has been built at the Tel-Aviv University,
which consists of a sample cell (Fig. 1) and a thermostat (Fig. 2). The cell is a thin-walled stainless cylindrical container (1 cm3 in volume) with an attached E-type thermocouple for temperature measurements (T), a miniature electrical heater and a lid. The thermostat contains one or two adiabatic thermal shields, while a differential E-type thermocouple connects the cell and the shields for regulating the temperature difference (dT) between them. In this calorimeter adiabatic conditions (zero heat exchange) are achieved by minimizing the heat exchange through heat conduction, convection and or radiation. The necessary conditions are thus attained by keeping the thermal shield at the same temperature as the cell. In practice, absence of heat loss is effected at a minimal (dT) at which the observed temperature drift (dT=dt) is close to zero and could be used as a correction factor in calculations of the specific heat. The automation of the calorimeter operation is accomplished by two PC-based, data-acquisition systems. The first system is intended to control the adiabatic conditions by supplying electrical power to the shield, and includes a two-channel HP 34420A nanovoltmeter (USA), which measures the voltage of the above-mentioned differential thermocouples. The nanovoltmeter sends data to a PC via HP-IB card and cable. The data are processed by the program, simulating two-channel PID controller, written with visual programming language}HP VEE. The controller outputs analog signal via D/A converter (a PC card), which drives the dual power amplifier connected to the heaters of both thermal shields. The second PC controls instruments and reads T2t data from an HP 3458A multimeter (USA) and the power of cell heater (P) from a K199 scanning multimeter. The heating power is supplied by a current
Fig. 1. Calorimetric cell with the specimen: (1) cell; (2) cover; (3) rolled amorphous ribbon specimen; (4) heater in cartridge; (5) thermocouple. For details see text (Materials and Methods).
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3. Results 3.1. Microscopic observations
Fig. 2. Adiabatic calorimeter: (1) calorimetric vessel; (2) adiabatic shield; (3) E-type thermocouples; (4) tubular heater; (5) specimen; (6) personal computer with GPIB interface. The miniature calorimetric vessel (1) is placed inside an adiabatic shield (2), and the whole adiabatic structure is mounted inside a vacuum chamber (not shown). The vessel and shield temperatures are monitored by E-type thermocouples (3). The heating is provided by a small tubular heater (4), which is inserted into the central hole of the specimen (5). All the measurement process control is carried out by a PC via a GPIB interface to programmable sources, A/D and D/A converters.
source. All procedures and controls during specific heat measurements are integrated in the HP VEE program. The collected data are processed, stored and displayed on the screen as various graphs. For calculation of specific heat values, Cp (J/K g) the following expression is used: Cp ¼ PðM * dT=dtÞ2A=M; where P is heating power of the cell heater (W), M is mass of the sample (g), dT=dt is the rate of heating, corrected to the temperature drift (K/s), and A is a heat capacity of the empty cell (J/K). The temperature rate (a quasiadiabatic heating regime) varied from 20 up to 70 K/h. Our measuring system in this range allowed precise temperature control of better than 0.01 K, which was enough for an accuracy in heat capacity and specific heat measurements of about 99%. Since the sample took 50% of the total cell heat capacity, the net heat capacity of the sample (after subtracting the value for the calibrated container) was measured to an accuracy of 2–3%.
Fig. 3a provides an overall view of the silk caps obtained via LM. In the natural state, these silk caps seal the cell outlets from underneath. At first they are of a cream color (Bar=1 cm). Above the caps, one can see the strata of the cell walls. Note that the silk making up the caps is the thickest of the whole weave, whereas the more interior part, that is, the part of the weave extending into the cell and comprising the sleeve is considerably thinner. In Fig. 3b, also taken via LM, one can see the silk caps after their rupture by the emerging imagines. The silk cap is now torn but its remnants are collectible, and indeed the worker hornets remove all traces of the silk cap before the queen oviposits again into the vacated cell. Fig. 3c, taken via SEM, shows a single silk fiber with grooves surrounding it, the latter comprising points devoid of the sericin proteins (the outer coat) and revealing the underlying fibroin proteins of the core fibril. Fig. 3d again shows a similar picture but at higher magnification, the intermittently absent sericin annuli now better visible. Fig. 4a, taken via SEM, shows entire fibers laid down in various directions and in various layers, and between them}the plaque-like silk plates of diverse geometric configurations. Fig. 4b shows more such plates and Fig. 4c, taken from the silk padding the interior of the cell, shows the plaque-like plates covering the entire inside surface, with the silk fibers criss-crossing the plates and appearing to be impressed into them. Fig. 4d, taken via TEM, shows a section through such a surface which contains a contribution of a number of fibers. What is evinced here is an array of fibers (fibrils?) displaying electron-dense areas and between them areas of a lighter color. This is usually the picture one sees wherever sections are made through the silk. 3.2. Thermoelectric measurements Fig. 5 provides a typical picture of a silk preparation taken from the interior silk padding of the cell. The preparation is contained within a Faraday cell which, in turn, is placed in an incubator sensitive to temperature increase in the dark (Litinetsky et al., 1998). Upon increase of the electric charge from 0 nA to about 90 nA, alongside a hike in the temperature between 208C and 338C, and at a RH above 90%, any further increase in temperature does not result in a further increase in electric charge. On the other hand, decrease of the temperature to 208C results in rapid drop of the charge down to 0 nA. After three repetitions of this procedure yielding the same results, we now introduce illumination (700 Lux white light), whereupon the charge is somewhat smaller while each cycle gradually lengthens, from
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Fig. 3. (a) The SEM sections of the silk caps of hornet pupae in a comb in their natural position. Bar=1 cm; (b) the same silk cap appearance after the eclosion of the imagines (adults) from the cell; (c) one single silk fiber with interruptions in the outer layer (sericin); (d) a single fiber with interruptions in the outer coat at higher magnification. For more details see text.
35 min in the dark to as much as 75 min in light, albeit it abbreviates again when the preparation is resubjected to darkness. Perhaps the prolongation of the cycles under illumination is due to changes, which are caused by the light, and the difficulty in cooling down to a basic level. When integrating the electrical charge obtained at every increase in current (in increase in temperature) one obtains an average of 200 mCoulomb (mC). 3.3. Specific heat measurements A total of about 0.5 g of silk was placed on six different occasions in a calorimeter to determine the specific heat of the silk caps. The results are shown in Fig. 6, in which: Fig. 6a represents runs 1 and 2 performed at a change rate of 70 K/h; Fig. 6b represents run 3 at 50 K/h; Fig. 6c represents run 4 at 20 K/h; Fig. 6d represents run 5 at 40 K/h; and Fig. 6e represents run 6 performed at a rate of 60 K/h. Finally, Fig. 6f represents a summation of all the six runs. As can be seen from this figure and all the preceding ones: (1) the silk did not undergo denaturation during any of the runs; (2) in each run one can discern three different segments, to wit: (a) from 308C to around 08C}a flat area with little changes; (b) from 08C to around
30–408C}a marked increase in the specific heat; and (c) from above 308C (or sometimes from above 408C) to around 70–808C}there are evident changes in the specific heat, comprising non-uniform increases and decreases. The curve of the generalized specific heat within the temperature range of 2–358C is given in Fig. 7, while that within the range of 20–458C is given in Fig. 8. A final parameter investigated was the change in weight upon temperature change, as shown in Fig. 9. As can be seen, the silk weight losses measured by thermogravimeter (TA Instruments, SDT 2960, England) within a temperature range of 20–808C showed a drop of about 7% and the drop continued also beyond this range albeit at a lesser extent. However, it is worth mentioning that when the silk preparation was left overnight in the laboratory it reverted to its initial weight and did not undergo denaturation, so that it could again be measured with the same basic levels.
4. Discussion So far as our heat capacity measurements are concerned, we draw attention to Fig. 6, which presents
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Fig. 4. (a) Silk fibers of a silk cap. Only a small area (right) is covered by a flat; (b) the silk coat in a section of the sleeve close to the silk cap. One sees separate fibers but most of the fibers are connected to the flats; (c) inside the cocoon the silk fibers are already embedded in the flats; (d) a TEM section of a silk fiber. One can see fibrils (electron-dense running along the silk fiber and between them there is some bright matter. Bar =1 mm). For more details consider text.
Fig. 5. Thermoelectric measurements on an area of a silk sleeve. Every cycle yields some 90–100 nA. The integral of the area includes some 200 mC.
several runs carried out at different rates. Note that at the low end of the range (268C to 58C) the specific heat remains rather unchanged and is roughly equal to 1.2 J/ g K. This is a value befitting many organic polymer
materials (Mark, 1996) and one to be expected for our vespan silk. However, from 58C onwards, the specific heat increases at more than a linear rate so that by about 408C it exceeds 2 J/g K, thus, exceeding the ‘normal’
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Fig. 6. Measurements of the specific heat of silk when measured at a different charge rate: 20, 40, 50, 60 and 70 K/h. Usually, one can see straight line (specific heat) between –30 till 58C, an increasing line between 5 till 30(40)8C and after 30(40)8C different steeply increasing specific heat and decreasing specific heat (as if this an area of phase transition).
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Fig. 7. Between 28C and 358C there is a curve of the generalized specific heat.
Fig. 8. The curve within the range of –208C to 408C. (For more details see text.) The area (integral) between points AB and AC represents the heat of desorption.
value by more than 50%. More precise measurements taken at slower rates evince even greater (80%) increase of the specific heat. Such a boost of the specific heat does not appear to be the result of any inner structural change in the silk since the very next run (after an overnight pause) roughly duplicates the initial measurements. Furthermore, a temperature of 408C is still far below that posing the risk of possible denaturation. A clue as to how to interpret this unexpected specific heat increase at elevated temperature may be gleaned from Fig. 9, which presents weight loss of our specimens
as a function of temperature. One can see here that the weight is systematically decreasing with increase in the temperature. This could account for the specific heat growth at ambient temperature of any material with a developed surface such as hornet silk. Obviously, also, desorption of water should take place in parallel to the heating. Since the calorimeter is a sealed apparatus, the desorption inside it leads to a respective rise in the humidity of the ambience just as it does in the natural nest. This process seems to be reversible, as seen in Fig. 6f, where the curves represent the specific heat of
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Fig. 9. The silk weight losses within a temperature range of 20–808C showed a drop of about 7%. This weight loss is reversible.
the same sample after a fortnightly interruption. Note that these curves are rather similar to that of the first run, which means that the water from the atmosphere has been adsorbed again to almost the same extent. We therefore suspect that a nycthemeral sorption–desorption cycle actually contributes to temperature regulation within the nests and comb cells of hornets. In line with such a conjecture, we believe that one could easily estimate the relevant heat of the adsorption–desorption process in the range of 0–408C. This heat can actually be calculated from an analytical presentation of the experimental data in Fig. 8. Thus, the total specific heat (averaged from two runs) of hornet silk can be derived by polynomial approximation from Cp0 ¼ 1:34 þ 1:25102 t þ 1:6104 t2 ;
J=K g;
ð1Þ
where t is the temperature in degrees of Celsius. The ‘regular’ part (extrapolated from the low-temperature curve) of it (actually the specific heat of dry hornet silk or silk with frozen water) is approximately expressed by Cp00 ¼ 1:23 þ 1:67103 t;
J=K g
ð2Þ
as for the area between the experimental curve (points and Eq. (1)) and the line of Eq. (2). Presumable additional heat of desorption (H in J/g) is, as usual, the area between the experimental curve (points and Eq. (1)), calculated by integrating the difference between
the two equations for CpðtÞ : Z 408C fCp0 Cp00 g dt ¼ 0:11t þ 5:5103 t2 H ¼ 0
þ 5:3105 t3 ;
J=g:
ð3Þ
In the chosen range of possible temperature desorption from 08C up to 408C the total possible desorption of heat is H ¼ 17 J/g. The corresponding amount of desorbed water can be roughly estimated from Fig. 9. This amount is determined to be M ¼ 0:011 0:003 g. As a result, one gets the heat of sorption L ffi 1545 420 J/g, or 370 100 cal/g of water. This is somewhat less than the latent heat of bulk water’s vaporization (539 cal/g). Such a value is rather usual for ad- or desorption heats of water at developed organic or non-organic surfaces. The water that is intermittently adsorbed and desorbed originates from the ambient air. The reason for the observed cyclicity of the process probably resides in the discrepancy between the optimal nest temperature, which is almost a fixed 298C throughout the day and night (Ishay and Ruttner, 1971) and the ambient (extra-nest) temperature, which at night is less than optimum and in the day is higher than optimum (Ishay, 1965). Additionally there is the need to maintain a high RH (above 90%) inside the nest. Consequently during the daytime, when the relative humidity of the air is low, considerable water (relatively speaking) is vaporized and thereby helps to cool the nest. Indeed, on warm days, we have observed hornets to ‘hang’ drops of water upon the
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silk caps and also perform ventilatory movements around the combs and up to the nest entrance (Ishay et al., 1967). By the end of the day the excess water is vaporized in the course of this ventilatory process and does not condense as dewdrops, which might promote putrefaction in the rather dark, warm and humid nest loaded with organic matter. At night, the amount of water in the air needed to provide high relative humidity is less than at daytime so that the implementation of water ventilation is obtained. The silk plaque-like surfaces, which contain longitudinal fibers (Fig. 4d), are capable, in microcapillary fashion, of conducting water of suitable temperature around the entire pupal cocoon. This water at night serves to warm the encased pupa, and likewise, via the thermoelectric (Seebeck) process, the electric energy stored during the daytime in the silk cap is transmitted at night as electric current to heat the upper parts of the pupa, that is, the ones at a distance from the silk cap. The transport of current to the pupal milieu and its distribution throughout the plaque-like silk surfaces is probably achieved through the gaps occurring along the length of the fibers, where the sericin coat is disrupted, enabling the current-conducting fibroin core to come in direct contact with the surrounding silk surfaces. In sum, we believe that the described hornet silk serves both a passive role in sealing the interior of the cell against parasites or predators and thus protecting the defenseless pupa, as well as an active role in thermoregulation of brood combs and the entire nest.
References Brunet, P.C.J., Coles, B.D., 1974. B. Tanned silks. Proc. Roy. Soc. London 187, 133–170. Chekhova, G.N., Goren, S.D., Krieger, J.H., Linsky, D., Lusternik, V., Panich, A.M., Semenov, A.R., Voronel, A., 2000. Specific heat and nuclear quadrupol resonance study of thiourea}hexachlorethane inclusion compound. Z. Naturforshung 55a, 335–338. Duncan, C.D., 1939. A Contribution to the Biology of North American Vespine Wasps. Stanford University Press, Stanford University, CA. Fahrenholz, L., Lamprecht, J., Schricker, B., 1992. Calorimetric investigations of the different castes of honey-bees, Apis mellifera carnica. J. Comp. Physiol. B-Biochem. Systemic Environ. Phys. 162 (2), 119–130. Graziano, G., Catanzano, F., Barone, G., 1998. Prediction of the heat capacity change on thermal denaturation of globular proteins. Thermochimica Acta 321 (1–2), 23–31. Harak, M., Lamprecht, J., Kuusik, A., 1996. Metabolic cost of ventilating movements in pupae of Tenebrio molitor and Galleria mellonella studied by direct calorimetry. Thermochimica Acta 276, 41–47.
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Ishay, J., 1965. Entwicklung und Aktivit.at im nest von Vespa orientalis. Deut. Entomol. Zeit. N. F. Band. 12 (4/5), 397–419. Ishay, J., 1975. Caste determination by social wasps. Cell size and building behavior. Animal Behav 23, 425–431. Ishay, J.S., Benshalom-Shimony, T., Ben-Shalom, A., Kristianpoller, N., 1992. Photovoltaic effects in the oriental hornet. J. Insect Physiol. 38 (1), 37–48. Ishay, J., Bytinski-Salz, H., Shulov, A., 1967. Contributions to the bionomics of the oriental hornet Vespa orientalis. Israel J. Entomol II, 45–106. Ishay, J.S., Kirshboim, S., 2000a. Grid formation in hornet cuticle induced by silk spun during pupation. Biomacromolecules 1 (2), 224–231. Ishay, J.S., Kirshboim, S., 2000b. Electric charging and charge release in hornet silk. J. Optoelectronics Adv. Mater. 2 (1), 83–90. Ishay, J., Ruttner, F., 1971. Die thermoregulation im Hornisennest. Zeit. v. Physiol. 72, 423–434. Jongebloed, W.L., Rosenzweig, E., Kalicharan, D., Want, J.J.H.v.d., Ishay, J.S., 1999. Ciliary hair cells and cuticular photoreception of the hornet Vespa orientalis as components of a gravity detecting system: an SEM/TEM investigation. J. Electron. Micros. 48 (1), 63–75. Kirshboim, S., Ishay, J.S., 2000. Silk produced by hornets: thermophotovoltaic properties}a review. Comp. Biochem. Physiol. A. 127 (1), 1–20. Kobashi, K., Oguni, M., 1997. Calorimetric finding of an anomaly of a Schottky type peculiar to solid solutions in a chlorocyclohexane}bromcyclohexane system. J. Phys. Chem. Solids 58 (5), 6695–6699. Litinetsky, L., Steinovitz, H., Ishay, J.S., 1998. Thermoelectric properties of hornet silk caps during different stages of pupation. Phys. Chem. Phys. Med. NMR 30 (1), 129–140. Mark, E., (Ed.) 1996. Physical Properties of Polymers. Handbook. AIP, NY. Miyahara-Mituyoshi, Taniguchi-Shin, Morichi-Toshiki, 1999. Effect of fat content on specific heat of meats. Animal Sci. J. 70(9), J184–J188. Rudall, K.M., Kenchington, W., 1971. Arthropod silks: the problem of fibrous proteins in animal tissues. Ann. Rev. Entomol. 16, 73–96. Sabbah, R., Ider, S., 1999. Thermodynamics of intermolecular and intramolecular bonds in three carboxypyridinic acids (picolinic, nicotinic and isonicotinic acids). Can. J. Chem. 77 (2), 249–257. Schmolz, E., Bru. ders, N., Schricker, B., Lamprecht, J., 1999. Direct calorimetric measurement of heat production rates in flying hornets (Vespa crabro; Hymenoptera). Thermochimica Acta 328, 3–8. Sverdlov, A., Litinetsky, L., Pertsis, V., Ishay, J.S., 2000. Thermophotovoltaic (TPV) properties of hornet cuticle as dependent on relative humidity. Phys. Chem. Phys. Med. NMR. 32, 57–66. Voronel, A.V., 1974. High-resolution specific heat measurement. Physica A 73 (1), 195–210. Voronel, A.V., Linsky, D., Kisliuk, A., Drislikh, S., 1988. Heat capacity and equilibration time near Tc of YBa2Cu3O7. Physica C 153, 1083–1088.
Hornet silk: thermophysical properties J.S. Ishaya,*, L. Litinetskya, V. Pertsis1, D. Linskyb, V. Lusternikb, A. Voronelb a
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 Ramat-Aviv, Israel Department of Physics and Astronomy, Sackler Faculty of Exact Sciences, Tel-Aviv University, 69978 Ramat-Aviv, Israel
b
Received 2 September 2000; accepted 3 February 2001
Abstract Thermoelectric measurements performed on strips of hornet silk have shown marked rise in the electric charge upon increase of temperature between 208C and 338C. The increase was dependent on the relative humidity (RH), occurring only at RH above 90%. In tests of heat capacity, hornet silk was found to possess a specific heat of over 2 J/g K within a temperature range of 5–408C. Observation carried out via transmission electron microscopy (TEM) on silk sections reveal that the silk bears lengthwise electron-dense (dark) stripes separated by light-colored stripes. We assume that in the warm daytime hours, hornet silk can longitudinally transport water that serves to raise the RH and to provide the vaporization needed for nest thermoregulation, whereas at night the very same silk provides an electric charge and longitudinally transfers current and heat. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Hornet silk; Specific heat; Thermoregulation; Calorimetry; Phase transition
1. Introduction The space comprising the nest of the Oriental hornet, Vespa orientalis (Hymenoptera, Vespinae), houses both the adult hornet population as well as combs bearing the brood. The comb brood cells contain larvae, which, upon maturation, commence pupating by weaving a silk cocoon around their body. At first, each such pre-pupal stage spins a silk cap that seals its comb cell outlet from below (the cells having been built upside-down). At this stage, the cell outlet is the only portal into the cell that has been left unbuilt, whereas all the walls as well as the roof (actually bottom) of the hexagonal comb cell have already been built ab initio (by the workers). After completing the silk cap, the pupating larva continues weaving a silk sleeve around itself so that ultimately it becomes entirely encased in a silk weave, comprised of two separable layers (Duncan, 1939). At closer inspec*Corresponding author. Tel.: +972-3-640-9138; fax: +9723-640-9113. E-mail address: [email protected] (J.S. Ishay). 1 Part of a Ph.D. Thesis to be submitted to the senate of Tel Aviv University.
tion, the silk weave is shown to be composed of a ‘stringy’ element}actually one elongated winding silk strand}and additionally also dense, plaque-like flats (Litinetsky et al., 1998; Ishay and Kirshboim, 2000a; Kirshboim and Ishay, 2000). The strings comprising the stringy element are each composed of a central fibril}the core}made up of fibroin proteins (Rudall and Kenchington, 1971; Brunet and Coles, 1974) and, a surrounding coat made up of sericin proteins that are arranged in transverse, with dispersed apparently disconnected annuli (Ishay and Kirshboim, 2000b). Once we have elucidated the manner in which thermoregulation is achieved in the hornet nest, we deemed it highly interesting to compare it to the specific heat of various inorganic and organic compounds including those in various living organisms, whose bodies are, in fact, made up of tissues comprised of organic matter. The literature boasts lately of numerous data on this subject. Inter alia Sabbah and Ider (1999) investigated, by various techniques the heat capacity of small organic molecular compounds (carboxypyridinic acid); Graziano et al. (1998) studied the heat capacity of globular proteins; Miyahara et al. (1999) determined the specific heat of chicken meat of variable fat content; and
0306-4565/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 1 ) 0 0 0 1 0 - 9
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Kobashi and Oguni (1997) investigated the phasetransition states in a chlorocyclohexane–bromocyclohexane system. The above investigations focused on materials, mostly on organic ones. In contrast, Harak et al. (1996) addressed the issues of the metabolic cost of entire organisms in terms of the number of calories spent by an insect when activating its wings or part of the body}in this case the insects Tenebrio molitor and Galleria mellonella; Fahrenholz et al. (1992) concentrated on calorimetric issues in various castes of the honeybee; and Schmolz et al. (1999) found interest in calorimetric measurements in flying hornets. As for ourselves, we have been investigating thermoregulation in nests of the Oriental hornet since 1967 (Ishay et al., 1967) and undertook to ascertain the role, which the silk produced by hornet larvae plays in this process. By now, we know that this silk is made of proteins which make up its fibrilar moiety, namely, fibroin proteins comprising the core and sericin proteins comprising the coat of each fibril. The silk of hornets has been shown to possess electric properties. Thus, upon exposure to light (blue or UV), the silk produces a voltage of several scores to several hundreds of millivolts (mV) (Ishay et al., 1992), while in the dark, the silk produces a current of several scores of nano amperes (nA). Both the current and voltage levels are dependent on the relative humidity (RH) and are optimal when the latter is maximal, that is, close to 100% (Sverdlov et al., 2000). The present study investigated the dependencies between the specific heat of the silk and its thermoelectric properties.
2. Materials and methods Pre-pupal silk caps were obtained from hornet brood combs collected from natural nests in the field. The silk was gathered as previously described (Ishay, 1975) and stored under refrigeration at 48C till used for the following purposes: (1) for observations via light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM), as previously described (Jongebloed et al., 1999); (2) for measuring the thermoelectric current, as previously described (Litinetsky et al., 1998); and (3) for thermophysical study geared to ascertain the specific heat of the silk caps. A large number of silk caps having a total weight of 0.524 g were packed into a preliminarily calibrated container. Specific heat of the silk sample was measured in the range of temperatures from 268C up to +808C. The most accurate values of specific heat and enthalpy of phase transition have been determined by adiabatic calorimeters (Voronel, 1974; Voronel et al., 1988; Chekhova et al., 2000). To this end, an adiabatic calorimeter has been built at the Tel-Aviv University,
which consists of a sample cell (Fig. 1) and a thermostat (Fig. 2). The cell is a thin-walled stainless cylindrical container (1 cm3 in volume) with an attached E-type thermocouple for temperature measurements (T), a miniature electrical heater and a lid. The thermostat contains one or two adiabatic thermal shields, while a differential E-type thermocouple connects the cell and the shields for regulating the temperature difference (dT) between them. In this calorimeter adiabatic conditions (zero heat exchange) are achieved by minimizing the heat exchange through heat conduction, convection and or radiation. The necessary conditions are thus attained by keeping the thermal shield at the same temperature as the cell. In practice, absence of heat loss is effected at a minimal (dT) at which the observed temperature drift (dT=dt) is close to zero and could be used as a correction factor in calculations of the specific heat. The automation of the calorimeter operation is accomplished by two PC-based, data-acquisition systems. The first system is intended to control the adiabatic conditions by supplying electrical power to the shield, and includes a two-channel HP 34420A nanovoltmeter (USA), which measures the voltage of the above-mentioned differential thermocouples. The nanovoltmeter sends data to a PC via HP-IB card and cable. The data are processed by the program, simulating two-channel PID controller, written with visual programming language}HP VEE. The controller outputs analog signal via D/A converter (a PC card), which drives the dual power amplifier connected to the heaters of both thermal shields. The second PC controls instruments and reads T2t data from an HP 3458A multimeter (USA) and the power of cell heater (P) from a K199 scanning multimeter. The heating power is supplied by a current
Fig. 1. Calorimetric cell with the specimen: (1) cell; (2) cover; (3) rolled amorphous ribbon specimen; (4) heater in cartridge; (5) thermocouple. For details see text (Materials and Methods).
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3. Results 3.1. Microscopic observations
Fig. 2. Adiabatic calorimeter: (1) calorimetric vessel; (2) adiabatic shield; (3) E-type thermocouples; (4) tubular heater; (5) specimen; (6) personal computer with GPIB interface. The miniature calorimetric vessel (1) is placed inside an adiabatic shield (2), and the whole adiabatic structure is mounted inside a vacuum chamber (not shown). The vessel and shield temperatures are monitored by E-type thermocouples (3). The heating is provided by a small tubular heater (4), which is inserted into the central hole of the specimen (5). All the measurement process control is carried out by a PC via a GPIB interface to programmable sources, A/D and D/A converters.
source. All procedures and controls during specific heat measurements are integrated in the HP VEE program. The collected data are processed, stored and displayed on the screen as various graphs. For calculation of specific heat values, Cp (J/K g) the following expression is used: Cp ¼ PðM * dT=dtÞ2A=M; where P is heating power of the cell heater (W), M is mass of the sample (g), dT=dt is the rate of heating, corrected to the temperature drift (K/s), and A is a heat capacity of the empty cell (J/K). The temperature rate (a quasiadiabatic heating regime) varied from 20 up to 70 K/h. Our measuring system in this range allowed precise temperature control of better than 0.01 K, which was enough for an accuracy in heat capacity and specific heat measurements of about 99%. Since the sample took 50% of the total cell heat capacity, the net heat capacity of the sample (after subtracting the value for the calibrated container) was measured to an accuracy of 2–3%.
Fig. 3a provides an overall view of the silk caps obtained via LM. In the natural state, these silk caps seal the cell outlets from underneath. At first they are of a cream color (Bar=1 cm). Above the caps, one can see the strata of the cell walls. Note that the silk making up the caps is the thickest of the whole weave, whereas the more interior part, that is, the part of the weave extending into the cell and comprising the sleeve is considerably thinner. In Fig. 3b, also taken via LM, one can see the silk caps after their rupture by the emerging imagines. The silk cap is now torn but its remnants are collectible, and indeed the worker hornets remove all traces of the silk cap before the queen oviposits again into the vacated cell. Fig. 3c, taken via SEM, shows a single silk fiber with grooves surrounding it, the latter comprising points devoid of the sericin proteins (the outer coat) and revealing the underlying fibroin proteins of the core fibril. Fig. 3d again shows a similar picture but at higher magnification, the intermittently absent sericin annuli now better visible. Fig. 4a, taken via SEM, shows entire fibers laid down in various directions and in various layers, and between them}the plaque-like silk plates of diverse geometric configurations. Fig. 4b shows more such plates and Fig. 4c, taken from the silk padding the interior of the cell, shows the plaque-like plates covering the entire inside surface, with the silk fibers criss-crossing the plates and appearing to be impressed into them. Fig. 4d, taken via TEM, shows a section through such a surface which contains a contribution of a number of fibers. What is evinced here is an array of fibers (fibrils?) displaying electron-dense areas and between them areas of a lighter color. This is usually the picture one sees wherever sections are made through the silk. 3.2. Thermoelectric measurements Fig. 5 provides a typical picture of a silk preparation taken from the interior silk padding of the cell. The preparation is contained within a Faraday cell which, in turn, is placed in an incubator sensitive to temperature increase in the dark (Litinetsky et al., 1998). Upon increase of the electric charge from 0 nA to about 90 nA, alongside a hike in the temperature between 208C and 338C, and at a RH above 90%, any further increase in temperature does not result in a further increase in electric charge. On the other hand, decrease of the temperature to 208C results in rapid drop of the charge down to 0 nA. After three repetitions of this procedure yielding the same results, we now introduce illumination (700 Lux white light), whereupon the charge is somewhat smaller while each cycle gradually lengthens, from
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Fig. 3. (a) The SEM sections of the silk caps of hornet pupae in a comb in their natural position. Bar=1 cm; (b) the same silk cap appearance after the eclosion of the imagines (adults) from the cell; (c) one single silk fiber with interruptions in the outer layer (sericin); (d) a single fiber with interruptions in the outer coat at higher magnification. For more details see text.
35 min in the dark to as much as 75 min in light, albeit it abbreviates again when the preparation is resubjected to darkness. Perhaps the prolongation of the cycles under illumination is due to changes, which are caused by the light, and the difficulty in cooling down to a basic level. When integrating the electrical charge obtained at every increase in current (in increase in temperature) one obtains an average of 200 mCoulomb (mC). 3.3. Specific heat measurements A total of about 0.5 g of silk was placed on six different occasions in a calorimeter to determine the specific heat of the silk caps. The results are shown in Fig. 6, in which: Fig. 6a represents runs 1 and 2 performed at a change rate of 70 K/h; Fig. 6b represents run 3 at 50 K/h; Fig. 6c represents run 4 at 20 K/h; Fig. 6d represents run 5 at 40 K/h; and Fig. 6e represents run 6 performed at a rate of 60 K/h. Finally, Fig. 6f represents a summation of all the six runs. As can be seen from this figure and all the preceding ones: (1) the silk did not undergo denaturation during any of the runs; (2) in each run one can discern three different segments, to wit: (a) from 308C to around 08C}a flat area with little changes; (b) from 08C to around
30–408C}a marked increase in the specific heat; and (c) from above 308C (or sometimes from above 408C) to around 70–808C}there are evident changes in the specific heat, comprising non-uniform increases and decreases. The curve of the generalized specific heat within the temperature range of 2–358C is given in Fig. 7, while that within the range of 20–458C is given in Fig. 8. A final parameter investigated was the change in weight upon temperature change, as shown in Fig. 9. As can be seen, the silk weight losses measured by thermogravimeter (TA Instruments, SDT 2960, England) within a temperature range of 20–808C showed a drop of about 7% and the drop continued also beyond this range albeit at a lesser extent. However, it is worth mentioning that when the silk preparation was left overnight in the laboratory it reverted to its initial weight and did not undergo denaturation, so that it could again be measured with the same basic levels.
4. Discussion So far as our heat capacity measurements are concerned, we draw attention to Fig. 6, which presents
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Fig. 4. (a) Silk fibers of a silk cap. Only a small area (right) is covered by a flat; (b) the silk coat in a section of the sleeve close to the silk cap. One sees separate fibers but most of the fibers are connected to the flats; (c) inside the cocoon the silk fibers are already embedded in the flats; (d) a TEM section of a silk fiber. One can see fibrils (electron-dense running along the silk fiber and between them there is some bright matter. Bar =1 mm). For more details consider text.
Fig. 5. Thermoelectric measurements on an area of a silk sleeve. Every cycle yields some 90–100 nA. The integral of the area includes some 200 mC.
several runs carried out at different rates. Note that at the low end of the range (268C to 58C) the specific heat remains rather unchanged and is roughly equal to 1.2 J/ g K. This is a value befitting many organic polymer
materials (Mark, 1996) and one to be expected for our vespan silk. However, from 58C onwards, the specific heat increases at more than a linear rate so that by about 408C it exceeds 2 J/g K, thus, exceeding the ‘normal’
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Fig. 6. Measurements of the specific heat of silk when measured at a different charge rate: 20, 40, 50, 60 and 70 K/h. Usually, one can see straight line (specific heat) between –30 till 58C, an increasing line between 5 till 30(40)8C and after 30(40)8C different steeply increasing specific heat and decreasing specific heat (as if this an area of phase transition).
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Fig. 7. Between 28C and 358C there is a curve of the generalized specific heat.
Fig. 8. The curve within the range of –208C to 408C. (For more details see text.) The area (integral) between points AB and AC represents the heat of desorption.
value by more than 50%. More precise measurements taken at slower rates evince even greater (80%) increase of the specific heat. Such a boost of the specific heat does not appear to be the result of any inner structural change in the silk since the very next run (after an overnight pause) roughly duplicates the initial measurements. Furthermore, a temperature of 408C is still far below that posing the risk of possible denaturation. A clue as to how to interpret this unexpected specific heat increase at elevated temperature may be gleaned from Fig. 9, which presents weight loss of our specimens
as a function of temperature. One can see here that the weight is systematically decreasing with increase in the temperature. This could account for the specific heat growth at ambient temperature of any material with a developed surface such as hornet silk. Obviously, also, desorption of water should take place in parallel to the heating. Since the calorimeter is a sealed apparatus, the desorption inside it leads to a respective rise in the humidity of the ambience just as it does in the natural nest. This process seems to be reversible, as seen in Fig. 6f, where the curves represent the specific heat of
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Fig. 9. The silk weight losses within a temperature range of 20–808C showed a drop of about 7%. This weight loss is reversible.
the same sample after a fortnightly interruption. Note that these curves are rather similar to that of the first run, which means that the water from the atmosphere has been adsorbed again to almost the same extent. We therefore suspect that a nycthemeral sorption–desorption cycle actually contributes to temperature regulation within the nests and comb cells of hornets. In line with such a conjecture, we believe that one could easily estimate the relevant heat of the adsorption–desorption process in the range of 0–408C. This heat can actually be calculated from an analytical presentation of the experimental data in Fig. 8. Thus, the total specific heat (averaged from two runs) of hornet silk can be derived by polynomial approximation from Cp0 ¼ 1:34 þ 1:25102 t þ 1:6104 t2 ;
J=K g;
ð1Þ
where t is the temperature in degrees of Celsius. The ‘regular’ part (extrapolated from the low-temperature curve) of it (actually the specific heat of dry hornet silk or silk with frozen water) is approximately expressed by Cp00 ¼ 1:23 þ 1:67103 t;
J=K g
ð2Þ
as for the area between the experimental curve (points and Eq. (1)) and the line of Eq. (2). Presumable additional heat of desorption (H in J/g) is, as usual, the area between the experimental curve (points and Eq. (1)), calculated by integrating the difference between
the two equations for CpðtÞ : Z 408C fCp0 Cp00 g dt ¼ 0:11t þ 5:5103 t2 H ¼ 0
þ 5:3105 t3 ;
J=g:
ð3Þ
In the chosen range of possible temperature desorption from 08C up to 408C the total possible desorption of heat is H ¼ 17 J/g. The corresponding amount of desorbed water can be roughly estimated from Fig. 9. This amount is determined to be M ¼ 0:011 0:003 g. As a result, one gets the heat of sorption L ffi 1545 420 J/g, or 370 100 cal/g of water. This is somewhat less than the latent heat of bulk water’s vaporization (539 cal/g). Such a value is rather usual for ad- or desorption heats of water at developed organic or non-organic surfaces. The water that is intermittently adsorbed and desorbed originates from the ambient air. The reason for the observed cyclicity of the process probably resides in the discrepancy between the optimal nest temperature, which is almost a fixed 298C throughout the day and night (Ishay and Ruttner, 1971) and the ambient (extra-nest) temperature, which at night is less than optimum and in the day is higher than optimum (Ishay, 1965). Additionally there is the need to maintain a high RH (above 90%) inside the nest. Consequently during the daytime, when the relative humidity of the air is low, considerable water (relatively speaking) is vaporized and thereby helps to cool the nest. Indeed, on warm days, we have observed hornets to ‘hang’ drops of water upon the
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silk caps and also perform ventilatory movements around the combs and up to the nest entrance (Ishay et al., 1967). By the end of the day the excess water is vaporized in the course of this ventilatory process and does not condense as dewdrops, which might promote putrefaction in the rather dark, warm and humid nest loaded with organic matter. At night, the amount of water in the air needed to provide high relative humidity is less than at daytime so that the implementation of water ventilation is obtained. The silk plaque-like surfaces, which contain longitudinal fibers (Fig. 4d), are capable, in microcapillary fashion, of conducting water of suitable temperature around the entire pupal cocoon. This water at night serves to warm the encased pupa, and likewise, via the thermoelectric (Seebeck) process, the electric energy stored during the daytime in the silk cap is transmitted at night as electric current to heat the upper parts of the pupa, that is, the ones at a distance from the silk cap. The transport of current to the pupal milieu and its distribution throughout the plaque-like silk surfaces is probably achieved through the gaps occurring along the length of the fibers, where the sericin coat is disrupted, enabling the current-conducting fibroin core to come in direct contact with the surrounding silk surfaces. In sum, we believe that the described hornet silk serves both a passive role in sealing the interior of the cell against parasites or predators and thus protecting the defenseless pupa, as well as an active role in thermoregulation of brood combs and the entire nest.
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Ishay, J., 1965. Entwicklung und Aktivit.at im nest von Vespa orientalis. Deut. Entomol. Zeit. N. F. Band. 12 (4/5), 397–419. Ishay, J., 1975. Caste determination by social wasps. Cell size and building behavior. Animal Behav 23, 425–431. Ishay, J.S., Benshalom-Shimony, T., Ben-Shalom, A., Kristianpoller, N., 1992. Photovoltaic effects in the oriental hornet. J. Insect Physiol. 38 (1), 37–48. Ishay, J., Bytinski-Salz, H., Shulov, A., 1967. Contributions to the bionomics of the oriental hornet Vespa orientalis. Israel J. Entomol II, 45–106. Ishay, J.S., Kirshboim, S., 2000a. Grid formation in hornet cuticle induced by silk spun during pupation. Biomacromolecules 1 (2), 224–231. Ishay, J.S., Kirshboim, S., 2000b. Electric charging and charge release in hornet silk. J. Optoelectronics Adv. Mater. 2 (1), 83–90. Ishay, J., Ruttner, F., 1971. Die thermoregulation im Hornisennest. Zeit. v. Physiol. 72, 423–434. Jongebloed, W.L., Rosenzweig, E., Kalicharan, D., Want, J.J.H.v.d., Ishay, J.S., 1999. Ciliary hair cells and cuticular photoreception of the hornet Vespa orientalis as components of a gravity detecting system: an SEM/TEM investigation. J. Electron. Micros. 48 (1), 63–75. Kirshboim, S., Ishay, J.S., 2000. Silk produced by hornets: thermophotovoltaic properties}a review. Comp. Biochem. Physiol. A. 127 (1), 1–20. Kobashi, K., Oguni, M., 1997. Calorimetric finding of an anomaly of a Schottky type peculiar to solid solutions in a chlorocyclohexane}bromcyclohexane system. J. Phys. Chem. Solids 58 (5), 6695–6699. Litinetsky, L., Steinovitz, H., Ishay, J.S., 1998. Thermoelectric properties of hornet silk caps during different stages of pupation. Phys. Chem. Phys. Med. NMR 30 (1), 129–140. Mark, E., (Ed.) 1996. Physical Properties of Polymers. Handbook. AIP, NY. Miyahara-Mituyoshi, Taniguchi-Shin, Morichi-Toshiki, 1999. Effect of fat content on specific heat of meats. Animal Sci. J. 70(9), J184–J188. Rudall, K.M., Kenchington, W., 1971. Arthropod silks: the problem of fibrous proteins in animal tissues. Ann. Rev. Entomol. 16, 73–96. Sabbah, R., Ider, S., 1999. Thermodynamics of intermolecular and intramolecular bonds in three carboxypyridinic acids (picolinic, nicotinic and isonicotinic acids). Can. J. Chem. 77 (2), 249–257. Schmolz, E., Bru. ders, N., Schricker, B., Lamprecht, J., 1999. Direct calorimetric measurement of heat production rates in flying hornets (Vespa crabro; Hymenoptera). Thermochimica Acta 328, 3–8. Sverdlov, A., Litinetsky, L., Pertsis, V., Ishay, J.S., 2000. Thermophotovoltaic (TPV) properties of hornet cuticle as dependent on relative humidity. Phys. Chem. Phys. Med. NMR. 32, 57–66. Voronel, A.V., 1974. High-resolution specific heat measurement. Physica A 73 (1), 195–210. Voronel, A.V., Linsky, D., Kisliuk, A., Drislikh, S., 1988. Heat capacity and equilibration time near Tc of YBa2Cu3O7. Physica C 153, 1083–1088.