ABSTRACT The cuticles of four species of isopods, the terrestrial species rmadillidium vulgare and Porcellio scaber, and the marine species Idotea montereyensis and Idotea resecata, were examined, focusing attention on whether or not there is a detectable lipid layer such as occurs in insects which might aid in water-proofing the integument of these animals. The rates of water loss over a range of temperatures were compared. A comparison of such rates with those of animals which had been treated with chloroform, a lipid solvent, is presented, and the results suggest that similar methods used in the past may lead to erroneous conclusions. No lipids were detectable in the cuticles with histochemical techniques. Basic structural similarities and differences in the cuticles of the four species are given, and the significance of these results are discussed. INTRODUCTION Several researchers have looked at the question of possible cuticular water proofing in terrestrial isopods, focusing their attention on the permeability properties of the integuments of the arthropods (Edney, 1951, 1954, 1968; Warburg, 1968; Bursell, 1955). Much of their work has been based on earlier studies and methods used for investigating the same phenomenon in insect cuticles (Beament, 1945, 1958, 1959; Wigglesworth, 1945, 1948; Holdgate, 1956; Mead-Briggs, 1956). The work on insects typically shows the presence of a critical transition temperature at which layers of waxy, lipid material in the cuticle deteriorate and the permeability of the integument dramatically increases, as evidenced by increasing rates of water loss from the animals at higher temperature ranges (Wigglesworth, 1918). Although such waxy layers have been reported present in the epicuticle of many of the insect species for which it was tested, the evidence for such layers in isopods is less con¬ vincing and the results have been subject to several conflicting interpretations (Bursell, 1955; Edney, 1957). Often the approach utilized has been to note changes in the rates of water loss in insects and isopods after lipids present have been removed by treatment with lipid solvents, but such evidence which only shows the resulting increase in the rate of transpiration does not seem substantial enough to credit water-retaining properties of the animals solely to the presence of lipids in their cuticles. Only a few observations have been reported on the actual substructure of the isopod cuticle (Bursell, 1955), although many generalized descriptions of the arthropod cuticle have been given (Richards, 1951; Dennel, 1961; Lockwood, 1967). A visual examination of the cuticles of several species may reveal characteristics which could account for any differences or similarities in the rates of water loss among these animals The presence of a water proofing layer could be tested histochemically. The purpose of this study was to re-examine some of the water-retaining properties of several species of isopods. To determine whether or not any such properties are inherent to isopods, suggesting they might be considered as a form of pre-adaptation for their invasion of land, species representing quite different habitats were compared. Armadillidium vulgare Latreille (1804) is a terrestrial species capable of withstanding dry conditions, and is typically found where a Mediterranean climate prevails. Porcellio scaber Latreille (180h) is found in more humid terrestrial environments (Miller, 1938). Idotea (Pentidotea) montereyensis Maloney (1933) is found on algae primarily in the intertidal regions, where it may face partially exposed conditions (Menzies, 1950), and Idotea (Pentidotea) resecata Stimpson (1857) occurs on Macrocystis and was studied to represent one of the truly marine species of isopods (North, 1971). The investigation was conducted in two parts. The rates of evaporative water loss were examined with each species for a series of temperatures to get an idea of their relative water retaining properties. An examination of the structure of the cuticle of each species was undertaken simultaneously to determine the basic characteristic of their cuticles and to note any significant differences between the species. Suspecting that waxy substance, or lipids, might be in part responsible for any permeability shown by the cuticles, the effects of a lipid solvent on the rates of transpira¬ tion and on the substructure of the cuticle were examined. MATERIALS and METHODS Animals: Specimens of the terrestrial isopods A. vulgare and P. scaber were collected from Carmel Valley and Pacific Grove, Monterey County, California. The marine I. montereyensis was obtained from Dillon Beach, Marin County, California, and from along the Pacific Grove coastline south of Monterey Bay, while I. resecata was collected from Monterey Bay in the vicinity of Hopkins Marine Station. Prior to the experiments, the terrestrial animals were kept in large containers at a humidity of 90% and fed on leaf litter and carrots. The marine species were retained in separate tanks with aerated sea water at ambient temperature, and either Macrocystis or Phyllospadix presented as food. All were used within a week of being collected. Water Loss: The rates of water loss over a range of temperatures were determined for each of the four species. At least ten animals were used for each data point, except in the experiments on 1. montereyensis where only eight and five animals were used at each temperature when determining the rates of dead and live animals. Only intermolt male animals of approximately the same size were used, with the following average weights: A. vulgare, 70 mg; P. scaber, 10.1 mg; L. montereyensis, 66.5 mg; and I. resecata, 79.h mg. The animals were first weighed individually on a Mettler balance and then placed in cylindrical containers sealed with mesh at both ends. The containers were set inside a dessicating chamber such that none were stacked on top of each other. The low humidity of the chambers was achieved by using Drierite (anhydrous calcium sulfate) and was checked during the experi¬ ment with cobalt chloride indicator papers (Solomon, 1915) to be less than 10%. The temperature of the dessicating chambers was maintained by placing them in a regulated temperature cabinet and the temperature was recorded during each experimental run. The temperatures generally used were 53 253 293 38, and 50 C. The animals were re-weighed at the end of an hour's exposure to such conditions, and the weight loss was recorded. The surface area of the experimental animals was calculated for each species by tracing the outline of the outspread animals onto graph paper using a dissecting microscope with a camera lucida attachment (Wigglesworth, 1945). The following average figures were used for each of the given species: A. vulgare, 1.5 cm ; P. scaber 1.38 cm ; I. montereyensis, 1.1 cm; and I. resecata, 1.83 cm". The rates of water loss were calibrated and expressed as mg/cm/ hr. To reduce the amount of extraneous surface water initially clinging to them,,the aquatic animals, both live and dead, were blotted with paper towel ing and dried slightly with an air stream in a uniform manner, before the initial weighing. This method was checked for its accuracy and uniformity by re-wetting and weighing the same animal several times. For experiments on dead animals, the isopods were killed by exposure to ammonia vapors for roughly fifteen minutes, and then used immediately to determine the rates of water loss (Bursell, 1950). In order to compare the effects of a lipid solvent on the cuticle of isopods and to test for the removal of such lipids, the animals were refluxed in chloroform for 2 hours (Bursell, 1955; Holdgate. 1956; Mead-Briggs, 1956; Shaw, 1955; Beament, 1955). After filtering off the chloroform while it was still warm, blotting the animals, and allowing the chloroform to evaporate until almost no odor could be detected (Mead-Briggs, 1956), the transpiration experiments were carried out as noted above. For comparison the rate of evaporation from a free-water surface was found by exposing a shallow vial,with known diameter and filled with water, in the same apparatus (Wigglesworth, 1915). Histochemistry: Thin hand-cut sections were made of the exoskeleton of each species. Sectioning was best achieved by first killing the animal, either by exposure to ammonia vapors or by quickly removing the head. The peraeopods were carefully removed, and the animal was frozen with dry ice. The sectioning was done quickly, using razor blades, under a dissecting microscope. Sections cut without first freezing were examined as well. Some of the sections were stained with methylene blue, Sudan III, and Sudan IV, the latter two stains being specific for lipids. The methods followed were those given by Pantin (1918) and Pearse (1968). Whole animals and sections were also stained for the presence of lipids by placing the material in a dessicator and subjecting them to the vapors of a dilution of a 2% solution of osmium tetroxide. Untreated animals, both normal and stained, were compared with those of the same species which had been treated with chloroform. Inter- specific comparisons of the cuticles were likewise made. Most of the descriptions and dimensions which are given refer to sections of the dorsal part of the thoracic segments of the exoskeleton, as those were the regions easiest to section and carefully examine. An additional proceedure called for immersing the whole animals in a 10% Chlorox solution, followed by 5% potassium hydroxide and a series of alcohol and xylene baths (Becker & Roudabush, 1912). The hard, calcified components of the exoskeleton were all that remained after such treatment; these were examined both as whole mounts and as sections. RESULT. Water loss: The rates of water loss from both live and dead animals of each species are plotted in Figs. 1-h. Note that there seems to be no significant difference between the live and dead animals of A.vulgare, P. scaber, and 1. resecata, suggesting that whatever water-retaining properties these isopods may have are not due to active mechanisms. Similar results have been reported by other investigators (Bursell, 1955; Edney, 1951) working with terrestrial species. The one exception, that of I. montereyensis, in which the live and dead animals have slightly different rates, shows the reverse of what might be expected for the live animals. The deviation is probably due to experimental error and the size of that particular sample. The rates of water loss for dead animals of all four species are shown together in Fig. 5. Note that while there seems to be no difference between the two terrestrial species and between the two aquatic species, the differences in the rates between these two groups of animals are great. The results are simplified and compared with the rates of evaporative water loss from a free-water surface in Fig. 6. The differences are what one might expect, since the aquatic forms are seldom faced with the problems of water shortage. Holdgate (1956) and Shaw (1955) have reported a similar situation with the rates for species of aquatic insects being higher than those of the terrestrial forms. The rates of water loss by A. vulgare which have been treated with chloroform are shown in Fig 7 together with the rates of live and dead animals of that species. Two types of dessication chambers were set up to determine whether weight loss was truly related to water loss or whether it might also represent the loss of chloroform - a heavier and more volatile substance than water. The first set was identical to those used in the previous experiments; the second set contained an open vial of chloroform which was used to saturate the chamber with chloroform vapors and insure that most of the weight loss recorded was actually due to water loss and not to the evaporation of chloroform. Note that the rates of the chloroform treated animals are generally higher and follow the same basic curve when compared to live and dead animals. The results also show that the rates of water loss which are recorded from animals following treatment with such a solvent can greatly be effected by the amounts of the solvent still remaining in the animals, as evidenced by the discrepency between the rates figured in which the two different sets of dessicating chambers were separately used. Histochemistry: The arthropod cuticle can be seen to have three main subdivisions. The thin, outer epicuticle is generally quite distinct from the inner exocuticle and endocuticle, which become very difficult to correctly distinguish in A. vulgare and P. scaber. Measurements of the thicknesses of each of these layers were made for each species, using a compound microscope with a bright field objective and a calibrated micrometer. The results are given in Table 1, along with a few values reported for the cuticle thickness of various insects. Cross sections of each species are shown simplified in Figs. 8. and 9. In all four species, the epicuticle is seen to be a very thin, almost transparent and non chitinous layer. It shows no birefringence when viewed with polarized light. There is no color reaction of the epicuticle itself with any of the applied stains. The surface texture of the species differed. That of P. scaber is notably irregular, particularily at the posterior region of the thoracic segments, as is shown in Fig. 10. The surfaces of A. vulgare and P. scaber are frequently covered with fine setae, which are not birefringent and which are most easily visible when cross sections are viewed under a phase contrast microscope. The surface structures of I. montereyensis and 1. resecata show irregularities as well, particularily on the epimeral plates. The epicuticle appears to have been laid down in sheet-like subunits, which are diagrammed in Fig. 11 as they are seen for a few species viewed under polarized light. Such structures remain after the treatment of the cuticles with chloroform. The exocuticle and endocuticle are oftern characterized by lamellae which run horizontal to the surface of the cuticle. Such lamellae were visible in all four species, but their thicknesses. uniformity, and numbers could not be ascertained. Pore and gland canals could be seen as well, though more easily in the terrestrial species. Using a phase contrast microscope, the gland canals could been seen to extend vertically through the entire cuticle, although they often seemed to be curly rather than extending straight. No branching of canals was seen in any of the species. With A. vulgare, small deposits of material were especially visible at the surface endings of the canals; such endings stained darkly with methylene blue and osmium tetroxide both before and after treatment of the animals with chloroform. Such surface deposits are not uniform throughout the cuticles, but rather are distributed in patches above regions of darkened material, presumably tanned protein substances. Theexocuticle and endocuticle of A. vulgare and P. scaber are almost transparent, can be seen best with a phase contrast micro¬ scope, and show birefringence under polarized light. The endocuticle of A, vulgare stained with methylene blue, which Schatz (1952) describes to be a reaction characteristic of several insect cuticles. Red pigmentation in the exocuticle, and sometimes a green coloration in the endocuticle, make the layers more distinguishable in the two marine species. Such pigmentation could not be effectively removed priof to staining by up to four days exposure to short ultraviolet light. There are also frequently packed, crystal-like amber structures in the region between the red exocuticle and the endocuticle in both of the aquatic species, as are shown in Fig. 9. Such structures fail to stain with any of the lipid stains and seem to be birefringent. I could not determine their structure or function. The changes in the cuticle after chloroform treatment are most noticeable in cross sections of A. vulgare. The basic, multi-layered structure remains, but the pore canals and lamellae are more visible, and it appears as if the canals may be wider. Of particular note is the amber or dark colored material which can be seen to fill the canals in the endocuticle to a line which is uniformly horizontal with the surface of the cuticle, as is shown in Fig. 12. Such a phenomenon is never visible in the normal, untreated cuticle, and it seems as if the material beneath the integument is being drawn up through the cuticle via the pore canals. Staining with Sudan III and IV shows no lipids in the cuticle, either before or after treatment with chloroform. There is considerable staining in areas just beneath the cuticle, even after chloroform treatment, indicating that lipids are present there and are not completely removed by the chloroform. DISCUSSION Whether or not there is a specific critical temperature at which the water proofing breaks down in isopod cuticles, such as those temperatures suggested by Bursell (1955) and others is still disputable (Edney, 1964, 1968; Lindqvist, 1972; Beament, 1958) and certainly the presence or suggestion of such points is not adequate criteria for the presence of a waxy layer which controls the permeability of the cuticles (Edney, 1957), although investigators in the past have frequently maintained such conclusions. The increased rates of water loss in the terrestrial isopods A. vulgare and P. scaber between the 30 - 50 C temperature range could reflect the breakdown or activation of some other structural component of the cuticle, thereby increasing the permeability of the integument. Most of the evidence points toward such a process as being passive. although recent experiments by Lindqvist (1972) using different approaches suggest that temperature effects on other body parts. such as the ailmentary canal and discharges by it, are perhaps more accountable for changes in the rates of water loss than are changes in the permeability of the integument. Lipids suspected to be active in the water proofing of the cuticle can be extracted with solvents such as chloroform and ether (Bursell, 1955; Richards and Korda, 1948). The rates of water loss after such treatment is usually found to be greater than the rates of untreated animals, as shown in this study using A. vulgare (Fig 7). Increases in the transpiration rate after a similar treatment have been reported by experimenters working with insects as well as isopods (Beament, 1945, 1955; Wigglesworth, 1945; Holdgate, 1956; Klinge, 1936; Umbach, 1934), but usually the increase in transpiration is shown for only one temperature point. When one graphs the rate of water loss versus temperature for a wide range of temperatures, the results show higher transpiration rates by the chloroform treated isopods, but the shape of the graph is almost identical with that which depicts the rates of untreated live and dead animals. The greater weight losses by chloroform treated animals which have been reported and interpreted to be due to increased water loss resulting from removal of lipid waterproofing and the subsequent greater permeability of the integument may in fact only reflect the loss of chloroform by these animals. To test this, dessicator chambers saturated with chloroform vapors were used, and the results indicated that it is indeed difficult to distinguish in such experiments the difference between water loss and the loss of the heavier compound, chloroform. Because of this, and the possibility that chloroform may have a variety of other effects on the structure of the whole animal, data which has been reported relative to chloroform treatment and increased transpiration rates seems questionable. Such greatly increased transpiration rates following solvent treat- ment may not be more than an artifact of the methods used. Cross sections of the cuticles reveal distinct differences among the four species examined, although there seem to be a few categorical differences between the marine and terrestrial forms. That A. vulgare has the thickest cuticle and is also the most terrestrial of the isopods studied seems significant. The irregularities in the surface texture and subunits could be an adaptation for the retention of water. No lipids were detected within the cuticle of any of the four species examined, although it is certain that there is a fatty layer beneath the tuticle in A. vulgare, P. scaber, and I. montereyensis. Perhaps water proofing is provided by only a small amount of lipid, such as would form a monolayer or be combined with other structural components of the cuticle, so as to not be detectable by simple histochemical methods. Perhaps the water-retaining properties of the cuticle are not at all dependent on the presence of any lipid. This study has shown several things with regard to the structure and function of the isopod cuticle. There is no pre-adaptation apparent in aquatic species when compared to the terrestrial isopods. It has not been possible to detect lipids in the cuticle using histochemical methods. Most importantly, the methods used, adopted from those cited in past insect and isopod literature, cannot separate a variety of possible explanations for the results which are obtained from such experiments. The structural effects of chloroform in of these animals are uncontrollable, as are the increases in the rates of measured "water loss", as such values are erroneously obtained and reflect a weight loss due to both water and solvent evaporation. REFERENCES Beament, J.W.L. 1945. The cuticular lipoids of insects. J. Exp. Biol. 21:115-131. . 1958. The effect of temperature on the water proofing mechanism of an insect. J. Exp. Biol. 35:194-519. 1959. The water-proofing mechanisms of arthropods. I. The effect of temperature on cuticle permeability in terrestrial insects and ticks. J. Exp. Biol. 36:391-122. Becker, E.R. and R.L. Roudabush. 1942. Brief Directions in Histological Technique. Lowa State College Press. Bursell, E. 1955. The transpiration of terrestrial isopods. J. Exp. Biol. 32:238-255. Dennel, R. 1961. The integument and exoskeleton, in The Physiology of Crustacea. T.H. Waterman, editor. Academic Press, New York. Holdgate, M.W. 1956. Transpiration through the cuticles of some aquatic insects. J. Exp. Biol. 33:107-118. Edney, E.B. 1951. The evaporation of water from woodlice and the millipede Glomeris. J. Exp. Biol. 28:91-115. 1954. Woodlice and the land habitat. Biol. Rev. 29:185-219. 1957. The Water Relations of Terrestrial Arthropods. Cambridge Univ. Press. . 1968. The transition from water to land in isopod Crustaceans. Am. Zocl. 8:309-326. Edney, E.B. and J.O. Spencer. 1955. Cutaneous respiration in woodlice. J. Exp. Biol. 32:256-269. Klinger. 1936. Arb. Phys. angew Ent. Berl. 3:16-69,115-151. (cf. Wigglesworth, 1915). Korschelt. 1923. Der Gelbrand, Dytiscus marginalis. I. Leipzig. (cf. Wiggles worth, 1918). Lindqvist, O.V., I. Salminen, and P.W. Winston. 1972. Hater content and water activity in the cuticle of terrestrial isopods. J. Exp. Biol. 56:h9-55. Lockwood, A.P.M. 1967. Aspects of the Physiology of Crustacea. W.H. Freeman & Co. San Francisco. Mead-Briggs, A.R. 1956. The effect of temperature upon the permeability to water of arthropod cuticles. J. Exp. Biol. 33:737-749. Menzies, R. 1950. The taxonomy, ecology, and distribution of northern Californian isopods in the genus Idothea with the description of a new species. Washmann J. Biol. 8(2):55-195. Miller, M.A. Comparative ecological studies on the terrestrial isopods of the San Francisco bay region. U.C. Publications in Zool. 13(7):113-112. North, W. J. 1771. The Biology of Giant Kelp Beds (lacrocystig) in California. Verlag Von J. Cramer. Pantin, C.F.A. 1948. Notes on Microscopial Techniques for Zoologists. Cambridge Univ. Press. Pearse, A.G.E. 1968. Histochemistry: Theoretical and Applied. 3rd edition. Little, Brown, & Co. Boston. Richards, A.G. 1951. The Integument of Arthropods. Minneapolis: Univ. of Minnesota Press. Richards, A.G. and Korda, F.H. 1948. Studies on arthropods. II. Electron microscope studies on extracted cuticle. Biol. Bull. 94:212-235. Schafz L 1952. The development and differentiation of arthropod procuticle: Staining. Ann. Ent. Soc. Am. 15:678-685. Shaw, J. 1955. The permeability and structure of the cuticle of the aquatic larva of Sialis tataria. J. Exp. Biol. 32:330-352. Solomon, M.E. 1945. The use of cobalt salts as indicators of humidity and moisture. Ann. Appl. Biol. 32:75-85. Umbach. 1934. Mitt. Forstwirt Forstwiss. 5:216-218. (cf. Wigglesworth, 1915). Waloff, N. 1941. The mechanisms of humidity reactions of terrestrial isopods. J. Exp. Biol. 18:115-135. Warburg, M.R. 1965. Water relations and internal body temperature of isopods from mesic and xeric habitats. Physiol. Zool. 38:99-109. 1968. Water loss in isopods. Crustaceana. 1h:39-14. 1968. Behavioral adaptations of terrestrial isopods. Am. Zool. 8:515-559. Wigglesworth, V.B. 1933. The physiology of the cuticle and of ecdysis in Rhodnius prolixis, etc. Quart. J. Micro. Sci. 76:269-318. 1945. Transpiration through the cuticle of Sts. J. Exp. Biol. 21:97-114. 1948. The insect cuticle. Biol. Rev. 23:408-45 1918. Te structure and deposition of the outicle in the adult nealworm Tenebrio molitor (Coleoptera). uart. J. Micr. Sci. (cited as in the press). FTGURE CAPTIONS Figure 1. Rates of evaporative water loss for Armadillidium vulgare, live versus dead animals. The rates of evaporative water loss are plotted against temperature. The temperatures used for the live animals were 50, 38; 28, 21, and 6°C. The temperatures used for the dead animals were 50, 30, 30, 25, and 5°C. Each point represents the average rate for a given sample, and the respective standard deviations are shown. Figure 2. Rates of evaporative water loss for Porcellio scaber, live versus dead animals. The rates of evaporative water loss are plotted against temperature. The temperatures used for the live animals were 50; 383 29, 2h, and 6°C. The temperatures used for the dead animals were 503 38, 293 25; and 6° C. Each point represents the average rate for a given sample, and the respective standard deviations are shown. Figure 3. Rates of evaporative water loss for Idotea montereyensis, live versus dead animals. The rates of evaporative water loss are plotted against temperature. The temperatures used for the live animals were 38, 293 253 and 6 C. The temperatures used for the dead animals were 50; 383 295 253 and 7°C. Each point represents the average rate for a given sample, and the respective standard deviations are shown. Figure l. Rates of evaporative water loss for Idotea resecata, live versus dead animals. The rates of evaporative water loss are plotted against temperature. The temperatures used for the live animals were 383 293 21, and 5'C. The temperatures used for the dead animals were 50, 38, 29; 24, and 5°C. Each point represents the average rate for a given sample, and the respective standard deviations are shown. Figure 5. Comparative rates of evaporative water loss for A. vulgare, P. scaber I. montereyensis, and I. resecata, dead animals. The rates of evapora- tive water loss for dead animals of all four species are plotted against temperature. Each point represents the average rate for a given sample, and the respective standard deviations are shown. Figure 6. Patterns of evaporative water loss for typical aquatic and terrestrial isopod species. The patterns are derived from Figs. 1-5, and compared to the pattern of water loss at similar temperatures from a water surface of known area. Figure 7. Effects of chloroform treatment on the rates of evaporative water loss. The rates of evaporative water loss are plotted against temperature. The dashed line represents animals which were treated with chloroform and dessicated in chambers which were partially saturated with chloroform vapors initially. The solid line connecting points from the other chloroform-treated animals represents animals which were dessicated under normal experimental conditions. The rates of evaporative water loss from untreated animals, both live and dead, and plotted also. Each point represents the average rate for given sample, and the respective standard deviations are shown. Relative thicknesses of the cuticles of isopods and insects. Table I. Figures are given in microns. The range given represents the variability found in ten animals of approximately the same size. Cross section views of the cuticle of two terrestrial species of Figure 8 isopods, A. yulgare and P. scaber. The following key applies. epi- epicuticle; exo- exocuticle; endo- endocuticle; p-pore or dermal canal; 1- lamellae; s-setae. Figure 9. Cross section views of the cuticle of two aquatic species of isopods, L. montereyensis and I. resecata. The following key applies: epi- epicuticle; exo - exocuticle; endo- endocuticle; p-pore or dermal canal; 1- lamellae; c- crystal-like substruces , identity uncertain. Figure 10. Irregularities in the surface and contour of the cuticle of P. scaber Diagrammed are the irregularities typical of the thoracic segments, Figure 11. Surface views of A. vulgare and I resecata, as seen with polarized light. Such substructures in the epicuticle or exocuticle differ with the terrestrial and aquatic forms. Figure 12. The effects of chloroform on the substructure of the cuticle of A. vulgare. 30 28 26 24 22 20 c 18 16 14 0 0 12 10 Armadillidium vulgare • ive animals o dead animals o 30 20 10 TEMPERATURE oC 40 50 30 28 26 24 22 20 18 5 16 014 12 Q10 1 8 10 Porcellio scaber o live animals o dead animals 30 20 TEMPERATURE 40 50 e 0 0 44 40 361 30 28 26 20 16 14 12 10 8 6 O dotea montereyensis olive animals odead animals 10 30 20 TEMPERATURE o 40 50 30 28 26 24 22 20 18 16 0 14 0 — 12 10 8 O dotea resecata e live animals O dead animals 30 10 20 TEMPERATURE 40 50 • Armadillidium vlgare O Porcellio scaber A looted montereyensis 30 A dotea resecata 28 26 24 22 20 18 2 16 14 12 Q 1O 8 4 2 10 0 30 20 TEMPERATÜRE 40 50 0 C O L 30 20 10 — terrestrial species — aquatt species — free-water surface 25 TEMPERATURE 50 44 40 32 28 26 24 22 20 18 16 14 12 10 2 0 Armadillidium vulgare o live animals o dead animals treated animals ACHC AC treated C animals 20 30 TEMPERATÜRE 10 40 exposed 50 SPECIES Armadillidium Vulgare Porcellio scaber Idotea montereyensis Idotea resecata SPECIES Periplanta (adult) Rhodnius (abdominal tergites of adult) (abdominal tergites of 5th instar nymph) Tenebrio (adult) Dytiscus larva ISOPODS EPÉCUTICLE EXOCUTICLE 2.4-8 u 15-60 n 8-48 u 1-10 8-16 u 21-10 1-8 u 16-18 u INSECTS SOURCE Richards & Anderson (1912) Wigglesworth (1933) Wiggl sworth (1918) Korschelt (1923) ENDOCUTICLE 10-176 n 20-50 u 16-80 24-96 u TOTAL THICKNESS 10 u 30 u 60 u u up to 370 20-10 u TOTAL THICKNESS 60-215 u 28-116 u 56-104 u 88-10 u Armadillidiumvulgare ++ tat- . . . . . :: ....:. . Porcellioscaber — . .4 ..... ... --- . ..... ..... ... . . . . . ... .. ........ . . Cted moiterepess 3 MLE Ltg P Tresecata doteg ta - s.l.. 0. — -- erdo ...... — — Porcellio scaber t ... . . . ... . ...... ...... Armadillidium vulgare * l . 3. * . . .. . .. . dotea resecata . .. . .. . * . .