- Email: [email protected]
The effects of temperature and moisture on survival capacity, cuticular permeability, hemolymph osmoregulation and metabolism in the scorpion, Centruroides hentzi (banks) (scorpiones, buthidae)
Camp. Biochem. Physiof.Vol. lOOA, No. 4, pp. 833-837, 1991
03~-9629/91 $3.00 + 0.00 Pergamon Press pit
Printed in Great Britain
THE EFFECTS OF TEMPERATURE AND MOISTURE ON SURVIVAL CAPACITY, CUTICULAR PERMEABILITY, HEMOLYMPH OSMOREGULATION AND METABOLISM IN THE SCORPION, CEiW’RUROIDES HENTZI (BANKS) (SCORPIONES, BUTHIDAE) FRED FUNZO Department
of Biology, University of Tampa. Tampa, FL 33606, U.S.A. Telephone: (813) 253-6206 (Received 25 March 1991)
Abstract-1. The temperature and water relations of Centruroides hentzi females were investigated. At I2 and 72% relative humidity (RH), the lower and upper LT% were -4.5 and 43.7”C, and -4.7 and 45.l”C, respectively. When exposed to high temperature stress, survivorship was significantly greater under mesic conditions. 2. Cuticular water loss was higher under xeric conditions (12% RH), ranging from 0.061 mg/cm2/hr at 30°C to 0.211 at 41°C. 3. Exposure to dry air (O-5% RH) resulted in a significant increase in hemolymph osmolality: from 441 to 688 mOsm over a 5 day period. 4. Mean oxygen consumption rates increased from 161.7 mm3/g/hr at 34°C to 541.6 at 44°C. ATPase activity was significantly higher in animals acclimated and tested at 35°C.
INTRODUCTION
The distribution, activity and survivorship of terrestrial anthropods are closely associated with their ability to withstand temperature and humidity stress while resisting evaporative water loss (EWL) (Edney, 1977; Punzo, 1989a,b). Although insects have received a great deal of attention (see reviews by Edney, 1977; Cloudsley-Thompson, 1973, there is significantly less information concerning the temperature and water relations of arachnids (Pulz, 1987). Most of the available arachnid literature concerns desert spiders and scorpions (Hadley, 1970, 1974; Warburg et al., 1980; Robertson et al., 1982; Pulz, 1987; Punzo, in press). Only a few studies have concerned themselves with the effects of temperature stress on mesic scorpions (Hadley, 1990). There is currently no data available on the thermal biology of the scorpion, Certtruroides hentzi (Banks), a common buthid associated with mesic as well as the more xeric sand pine-scrub habitats of south-central Florida. Experiments were conducted in order to determine upper (ULTSO) and lower (LLT5,,) lethal temperatures, the combined effects of temperature humidity (RH) on survival capacity,
and
relative
cuticuiar permeability (measured as EWL), and osmoregulation, the effects of temperature on oxygen consumption rates, and the effects of thermal acclimation on ATPase activity. MATERIALS AND METHODS
Adult C. hen& were collected from Hillsborough and Orange Counties, Florida, during the spring and summer months of 1989 and 1990. Specimens were collected using pitfall traps. Since most of the animals collected during this sampling period were adult females (32-42 mm,
0.241-0.473 g), temperature experiments were confined to this sex and life cycle stage. Scorpions were returned to the laboratory and maintained at 21°C and 70% RH under a 12L: 12D photo~riod regime. They were fed on a diet of crickets and mealworm larvae and provided with water on a weekly basis. Food and water were removed 48 hr prior to testing. Test temperatures were obtained through the use of a Precision Model 816 incubator (Chicago, IL). Relative humidity values within the incubator were obtained through the use of silica gel or saturated salt solutions as described by Winston and Bates (1960): silica gel (O-S%), LiCl(12%), NaCl (73 i 2%). All experiments were conducted under conditions of total darkness (Punt0 and Mutchmor. 1980). Lethal temperature and survival capacity determinarions Values for LLT% and ULT, were obtained according to the procedure described by Punzo and Huff (1989). To summarize, 10 scorpions were placed individually in glass containers. Four replicates of 10 animals yielded data on a total of 40 subjects for each experimental condition (RH values of 12 and 72%). For LLTw determinations, scorpions were exposed to low temperatures ranging from - 1.O to - 8.O”C at O.l”C intervals for a period of 1 hr under each condition of RH. Following the exposure interval, scorpions were removed from the incubator and returned to normal rearing conditions. The number surviving after 24 hr was recorded and expressed as per cent survival. Similar procedures were employed for ULTsO determinations. Scorpions were exposed to test temperatures ranging from 39 to 48°C at O.l”C intervals for a period of 1 hr under mesic (72% RH) and xeric (12%) conditions. These data were used to determine lethal temperatures (Table 1) as well as to assess the combined effect of low (LTS) and high (HTS) temperature stress and RH on survival capacity (Table 2). Evaporative water loss (EWL)/cuticular
permeability
All experiments were conducted in a desiccation apparatus previously described by Punzo and Jellies (1983). Air was recycled at a flow rate of 3.8 l/min and RH values (12 and 72%) were obtained as described above. Test 833
834
FREDPUNZO
Table 1. Mean upper (ULT& and lower (LLT~)lethal temperatures for adult female Cenrruroides hentri under xeric (12% RH) and mesic (72% RH) conditions (N = 40). One hour period of exposure. Values in parentheses represent +SE Relative humidity (%)
Test
Lethal temperatures (“C)
12 72 P’
ULT
LLT
43.7 (0.13) 45.1 (0.021 O.bl ’
-4.5 (0.05) -4.7 (0.04) NS ’
*Level of significance computed using Student’s r-test (Sokal and Rohlf, 1981). temperatures ranging from 30 to 41’C (Table 3) were used throughout these experiments. Air was bubbled through 5OOml of the saturated salt solution and then passed through cotton traps before entering the desiccation chamber housing the animals. Scorpions were placed individually in glass containers (five animals per replicate,
N = 25 for each test temperature) provided with small holes for air circulation. Animals were weighed individually on a Metler Analytical Balance to the nearest 0.005 mg at 1 hr intervals over a 24 hr period. EWL (cuticular permeability) was expressed as water (weight) loss (mg/cm2/hr) as described by Hadley (1990). The surface area of individual scorpions was obtained using the relationship, S = kW2”, where S represents surface area (cm2), W the body weight (mg), and k a constant (Edney, 1977). To obtain a value for k, scorpions of known weight were cleared in KOH. Their exoskeletons were spread over a grid paper containing 0.25 mm squares and measured with a Unitron H60 dissecting microscope. The k value obtained for adult females was 0.194 cm*/mg + 0.05. Hemolymph osmolality determinations
Hemolymph samples were obtained from each scorpion according to the method described by Punzo (1983). An incision was made in the abdominal region of CO,-anesthetized animals and hemolymph samples were collected in heparinized 1.0 ~1 pipettes. These pipettes were sealed at both ends with vasoline and frozen at - 30°C for subsequent osmolality determinations. Hemolymph samples were centrifuged for 20 min at 1000 rpm. Analyses were determined for 30 different scorpions exposed to temperature of 35°C at @5% and 72% RH in order to assess the influence of desiccating conditions on osmoregulation in this species Table 2. The effects of low (LTS) and high (HTS) temperature stress on the per cent survival of Centruroides henrzi females under xeric (12% RH) and mesic (72% RH) conditions (N = 40). One hour exposure period Relative humidity (%) 12
72
LTS (“C)
Per cent survival (%)
-6.0 - 5.0 -4.0 - 3.0 -2.0
33 63 60 73
-6.0 -5.0 -40 -3.0 -2.0
38 60 60 70
0
0
Table 3. The effects of temperature and relative humidity (RH) on cuticular permeability in Cenrruroides henrzi females after a 24 hr period. Cuticular permeability expressed as mean water (weight) loss in malcm’lhr (N = 251. Values in oarentheses noresent f SE
HTS (“0
Per cent survival (%)
39 40 41 42 43 44 45
98 95 80 63 55 10 0
39 40 41 42 43 44 45 46 47 48
95 95 93 83 73 63 50 25 5 0
A Wilcoxon Signed Rank Test (Sokal and Rohlf, 1981) showed the effects of RH (12 vs 72%) on per cent survival to be significant only under conditions of HTS (Z = 3.77, P < 0.01).
Water loss (mg/cm*/hr) Relative humidity (%) 12% RH 72% RH
temperature (“C) 30 31 32 33 34 35 36 37 38 39
0.061 0.062 0.067 0.073 0.083 0.092 0.137 0.161 0.167 0.175 0.184 0.21 I
40
41
(0.012) (0.011) (0.013) (0.010) (0.015) (0.01 I) (0.014) (0.021) (0.018) (0.016) (0.023j (0.025)
0.060 (0.012)
0.061 0.061 0.062 0.066 0.077 0.079 0.091 0.104 0.113 0.121 0.127
(0.014) (0.01 I) (0.013) (0.012) (0.014) (0.01 I) (0.009) (0.015) (0.008) io.011 j (0.013)
An ANOVA (Sokal and Rohlf, 1981) showed a significant effect of RH on cuticular permeability (F = 2 I .04, P < 0.001).
(Table 4). Measurements were taken over a 5 day exposure period. Hemolymph osmolality was determined using a Knauer osmometer and expressed as mOsm/g. Oxygen consumption determination and the effects of thermal acclimation on ATPase activity Experiments were also conducted in order to assess the effects of thermal acclimation at the enzymic (molecular) level in order to more fully understand the effect of temperature on metabolic processes in this species. ATPase activity was chosen owing to its overall significance in locomotor activity in arthropods. Scorpions (N = 40) were acclimated at either 15 or 35°C (Table 5). Homogenates were prepared using cephalothoracic tissue from C. hentzi females. Tissues from five scorpions were pooled for each test. Tissues were homogenized in a Frey Model 70 tissue homogenizer at 0°C with deionized water. ATPase determinations were conducted according to the method described by Anderson and Mutchmor (1971) and recorded as pg P/mg/animal/min. Phosphorus determinations were obtained with a Coleman Model 24 spectrophotometer at 600 urn and compared to reaction blanks (Punzo and Huff, 1989). The effects of temperature on metabolic rate (oxygen consumption) was also determined and expressed as mm’/g/hr as described by Hadley and Hill (1969). Oxygen consumption was measured using a constant pressure respirometer as described by Engelmann (1963). Forty scorpions were placed individually inside the respirometer. The apparatus was then sealed with stopcock grease and submerged in a Tectronic water bath (Chicago, IL). Oxygen consumption was measured over a temperature range of 34 to 44°C at 2.O”C intervals (Table 6). Readings were recorded every 30 min over a 24 hr period. Scorpions were weighed Table 4. Hemolympb osmolality (mOsm/g i SE) in Centruroideshenlzi females exposed to a temperature of 35°C under xeric and mesic conditions (N = 30 for each test condition). Measurements were taken over a 5 day exposure period. Values expressed as means * SE
Exposure (davs) I
2 3 4 5
Hemolymph osmolality (mOsm/g + SE) Relative humidity 72% &5% 441*21 504 * 33 521 f 47 601 f 39 678+44
422 i I7 431 i 29 439 i 37 443&21 461 + 35
An ANOVA (Sokal and Rohlf, 1981) showed a signilicant effect of relative humidity (F = 34.17. P < 0.01) on hemolymph osmoregulation.
Temperature and water relations in the scorpion Table 5. ATPase activity hg P/mg/min) in cold (15°C) and warm (35°C) acclimated Cenfruroides henrzi females. Values expressed as means f 1.0 SD (N = 40 for each experimental condition) Acclimation temperature (“C) 15 15 35 35
Test temperature (“C) 15 35 35 15
‘Level of significance computed (Sokal and Roblf, 1981).
ATPase activity 0.367 0.396 0.892 0.514
+ f + f
0.03 0.02 0.1 I* 0.07
using Student’s f-test, P < 0.01
before each test and body volumes determined by water displacement (0.01 ml) after testing. The respirometer allowed for only minimal movement by test animals. RESULTS The upper and lower lethal temperatures for C. henrzi are shown in Table 1. This scorpion can withstand significantly higher ambient temperatures under mesic conditions (451°C). When exposed to dry air, the ULTso value dropped to 43.7”C. Relative humidity had no similar effect on LLTso values. Previous studies have shown that ULTS for scorpions can range from 45 to 48°C (Hadley, 1990), and for some species can even exceed 50°C for short exposure periods. With respect to survival at low temperatures, body temperatures between - 11.9 and - 5.8”C have been reported for some higher altitude species (Hadley, 1974, 1990). The combined effects of low and high temperature stress on survival capacity as a function of RH are shown in Table 2. Under conditions of LTS, RH had no significant effect on survival rates. When exposed to HTS (4144°C) however, survival capacity was higher under mesic conditions. In dry air, 90% of the scorpions died when exposed to 44°C. At this temperature mortality decreased to 37% under mesic conditions. This is in general agreement with results reported for other mesic scorpions (Warburg et al., 1980; Warburg and Ben-Hoi-in, 1981; Robertson et al., 1982; Hadley, 1990) and suggests that cooling via EWL is minimal in these species. This is in contrast to results reported for mesic and xeric insects which have demonstrated the ability to withstand higher temperatures under dry air conditions due to effective evaporative cooling mechanisms for short periods of time (CloudsleyThompson, 1975; Punzo and Mutchmor, 1980; Punzo and Huff, 1989; Punzo, 1989b, in press). The effects of temperature and RH on EWL (cuticular permeability) are shown in Table 3. Water loss rates were significantly higher under xeric Table 6. Relationahip between air temperature and mean oxygen consumption rates in Centruroides hentzi females (N = 40). Values in parentheses represent *SE Temperature (“C) 34 36 38 40 42 44
Mean oxygen consumption rate (mm’lglhr) 161.7 180.2 239.3 277.8 312.5 541.6
(14.4) (12.9) (21.6) (18.9) (17.8) (41.5)
on metabolic
conditions over the entire range of test temperatures. This, in conjunction with the results reported above for lethal temperatures and survival capacity, indicate that C. hentzi, normally associated with mesic habitats in Florida, is not particularly well adapted to dry air conditions. These water loss rates are in general agreement with those reported for other mesic and xeric scorpions such as Scorpio maurus (Scorpionidae), Nebo hierochonticus (Diplocentridae), Buthotus judaicus and Leiurus quinquestriatus (Buthidae) (Warburg et al., 1980) as well as Parabuthus villosus (Buthidae) and Opisthophthalmus cupensis (Scorpionidae) (Robertson et al., 1982). Thus, it would appear that many scorpions minimize EWL under xeric conditions through behavioral mechanisms such as fossoriality and nocturnal activity patterns (Hadley, 1990) which are characterized by more favorable microhabitat conditions. The results for hemolymph osmolality following a 5 day period of exposure to combinations of temperature and RH are shown in Table 4. Exposure to dry air resulted in an increase in hemolymph osmolality as compared to results obtained in moist air. Previous studies on several different species of scorpions have also reported an increase in hemolymph osmolality under experimental dehydration (Warburg et al., 1980; Robertson et al., 1982; Burton, 1984; Riddle, 1985). Similar results have been reported for insects (Edney, 1977; Punzo, 1989b) and spiders (Pulz, 1987) as well, although some species are osmoregulators rather than osmoconformers. The effects of thermal acclimation on ATPase activity are shown in Table 5. There was no significant effect of temperature in animals acclimated at 15°C and tested at 15 or 35°C. Only in animals acclimated at 35°C was a significant effect found. The increase in ATPase activity at higher temperatures indicates some capacity for molecular (enzymic) compensation to changing ambient temperatures in this species. Although this type of response is common in some insects (Anderson and Mutchmor, 1971; Punzo and Huff, 1989) it has not been generally associated with arachnids (Pulz, 1987; Punzo, in press). Changes in ATPase activity can influence locomotor activity and have obvious adaptive consequences for foraging, mating, habitat selection and escape responses. The relationship between air temperature and oxygen consumption rates (metabolism) is shown in Table 6. Increases in temperature resulted in a concomitant increase in metabolic rate. This is most pronounced at the 4244°C interval and agrees with similar studies on other terrestrial arthropods which have shown a more significant increase in oxygen consumption rates as upper lethal temperatures are approached (Hadley and Hill, 1969; CloudsleyThompson, 1975; Pulz, 1987). DISCUSSION
An ANOVA (Sokal and Rohlf, 1981)showed a significant effect of temperature (F = 41.04, P < 0.001).
835
rate
Centruroides hentzi is commonly associated with mesic habitats in Florida characterized by sandy substrates. It can also be found in less common habitats such as sand pine and scrub associations where more xeric conditions prevail. This scorpion can reduce its exposure to unfavorable environmental
836
FREDPUNZO
conditions through burrowing and nocturnal activity patterns which represent behavioral mechanisms of thermoregulation commonly associated with scorpions (Cloudsley-Thompson, 1975; Edney, 1977). Although these adaptive responses are especially evident in desert-dwelling species, they are rather common in mesic species as well (Hadley, 1990). The upper lethal temperatures for C. hentzi females (Table 1) are similar to those reported for other mesic and xeric scorpions (Warburg et al., 1980; Warburg and Ben-Horin, 1981; Robertson et al., 1982) and tend to be somewhat higher than those reported for other arachnids and insects found in similar habitats (Punzo and Jellies, 1983; Punzo, 1989b, 1991; Hadley, 1990). It is important to keep in mind that seasonal acclimatization effects have been reported to alter lethal temperatures in some scorpions (Riddle, 1985) and other arthropods (Cloudsley-Thompson, 1975; Punzo and Mutchmor, 1980; Punzo and Huff, 1989). It would be interesting to determine lethal temperatures for C. hentzi collected during different months of the year (fall and winter) and compare them to the values presented in this study for animals collected during spring and summer months. The data on survivorship as a function of temperature and RH stress (Table 2) indicate that C. henlzi females are more resistant to HTS under mesic conditions. This is supported by the preference for nocturnal activity exhibited by this species (personal observation) as well as its biogegraphical distribution and microhabitat preferences. The survival of terrestrial arthropods is predicated upon their ability to limit desiccation through EWL. Transpiratory water loss (cuticular and respiratory) is usually enhanced under the dry air conditions associated with xeric habitats. For this reason, many xeric arthropods are characterized by a desiccationand anatomical structures resistant epicuticle designed to close body cavities opening at the surface, as well as appropriate behavioral strategies 1974; Cloudsley-Thompson, 1975; (Hadley, Warburg, 1986; Pulz, 1987; Punzo, in press). EWL is also facilitated by the relatively small body size and concomitant large surface area to volume ratio associated with arthropods. As a group, scorpions possess an integument highly impermeable to water (Hadley, 1990) that is most highly developed in xeric species. Cuticular permeability (water loss rates) in C. hentzi (Table 3) is significantly higher when animals are exposed to dry air conditions. Previous studies have shown that water loss rates in other scorpions exposed to an air temperature of 30°C can range from 0.025 to 0.049 mg/cm*/hr for xeric species, and from 0.064 to 0.270 mg/cm2/hr for species from more mesic habitats (Warburg et al., 1980; Robertson et al., 1982; Hadley, 1990). In C. hentzi, water loss rates at 30°C were 0.061 and O.O60mg/cm*/hr under xeric and mesic conditions, respectively. This is in agreement with the range of values mentioned previously for mesic scorpions. Previous investigators have also shown that over the temperature range used in this study, most of the EWL can be attributed to cuticular loss rather than respiration or excretion (Warburg et al., 1980). Where collecting conditions permit, future
studies should be conducted to analyse the relationship between temperature, RH and EWL rates for various life cycle stages (body size) for both sexes of C. hentzi. High temperature stress combined with exposure to dry air can lead to pronounced transitory water deficits in terrestrial arthropods regardless of behavioral and physiological mechanisms for reducing water loss. Such water loss can be accompanied by marked changes in hemolymph volume and osmolality (Riddle, 1985). Many insects have shown the ability to regulate the osmotic pressure of hemolymph despite the reduction of hemolymph volume normally associated with dehydration (Edney, 1977) whereas most scorpions have demonstrated the capacity to tolerate increasing hemolymph osmolality associated with water loss (Hadley, 1970, 1990). Warburg et al. (1980) reported a significant increase in hemolymph osmolality for several scorpions exposed to dry air conditions over a 7 day period. Furthermore, hemolymph osmolality increased to a greater extent in xeric species. In the present study, there was a significant increase in hemolymph osmolality when C. hentzi females were exposed to desiccating conditions (Table 4). At O--5% RH, individuals of this species were able to withstand an increase from 441 to 678 mOsm/g indicating that this species, like most scorpions, is an osmoconformer. One exception to this was reported by Robertson et al. (1982) for Parabuthus villosus, a large, non-burrowing scorpion inhabiting very arid regions of Ethiopia and frequently active during the day. This species exhibited the capacity for efficient ionic and osmotic regulation that compared with that demonstrated for some desert insects. More information is needed to assess the mechanisms responsible for osmoregulation in P. viflosus. The metabolic rates determined for C. hentzi (Table 6) are comparable to those reported for other scorpions (Hadley and Hill, 1969; Robertson et al., 1982), and are generally lower than values reported for insects (Punzo, 1989b). ATPase activity showed an acclimatory response at 35°C (Table 5). Presumably, this indicates a relationship between change in temperature and locomotor activity at the enzymic level associated with enzyme-substrate affinity allowing for maximal movement over a range of ambient temperatures. This will require further investigation in light of the fact that previous studies have reported the absence of a thermal acclimatory response for oxygen consumption rates in scorpions (Robertson et al., 1982) and other arachnids (Pulz, 1987; Punzo, 1991). This has been attributed to the markedly lower metabolic rates exhibited by spiders and scorpions. However, Riddle (1979) reported that high temperature acclimation results in a lateral shift in the metabolic ratetemperature curve in the desert scorpion, Paruroctonus utahensis. Acknowledgemenfs-I wish to thank Mr T. Punzo who assisted in the collection of specimens, and Dr Brian Garman, Department of Mathematics, who provided valuable assistance in the statistical analyses. A Faculty Development Grant from the University of Tampa made much of this work possible.
Temperature and water relations in the scorpion REFERENCES Anderson R. L. and Mutchmor J. A. (1971) Temperature acclimation in Tribolium and Musca at locomotory, metabolic and enzymic levels. J. Insect Physiol. 17, 22052219. Burton R. F. (1984) Hemolymph composition in spiders and scorpions. Comp. Biochem. Physiol. 78A, 613-616. Cloudsley-Thompson J. L. (1975) Adaptations of Arthropoda to arid environments. A. Rev. Entomol. 20, 261-283. Edney E. B. (1977) Water Balance in Land Arthropods. Springer, Berlin. Engelmann M. D. (1963) A constant pressure respirometer for small arthropods. Entomol. News 74, 181-187. Hadley N. F. (1970) Water relations of the desert scorpion, Hadrurus arizonensis. J. exp. Biol. 53, 547-558.
Hadley N. F. (1974) Adaptational biology of desert scorpions. J. Arachnol. 2, 11-23. Hadley N. F. (1990) Environmental physiology. In The Biology of Scorpions (Edited by Polis G. A.), pp. 321-340. Stanford University Press, Stanford, California. Hadley N. F. and Hill R. D. (1969) Oxygen consumption of the scorpion Centruroides sculpturatus. Comp. Biochem. Physiol. -29, 217-226.
_
Pulz R. (1987) Thermal and water relations. In Ecoohvsiology oj Spiders (Edited by Nentwig W.), pp.’ 2655. Springer, New York. Punzo F. (1983) Hemolymph chemistry of the spiders, Heteropoda venatoria (Sparassidae), Pisaurina mira (Pisauridae) and Amaurobius bennerti (Amaurobiidae). Comp. Biochem. Physiol. 75A, 647652.
Punzo F. (1989a) Composition of the hemolymph of mygalomorph spiders (Orthognatha). Comp. Biochem. Physiol. 93A, 757-760.
Punzo F. (1989b) Comparative temperature and water relations and hemolymph osmoregulation in the desert insects, Taeniopoda eques and- Schistocerca vaga (Orthoptera, Acrididae). Comp. Biochem. Physiol. 93A, 751-755.
837
Punzo F. Intraspecific variation to thermal stress in the tarantula, Dugesiellu echina Chamberlin (Orthognatha, Theraphosidae). Bull. Br. Arachnol. Sot. (in press). Punzo F. and Huff G. (1989) Comparative temperature and water relations and the effects of thermal acclimation on Tenebrio molitor and Tenebrio obscurus (Coleoptera: Tenebrionidae). Comp. Biochem. Physiol. 93A, 527-533. Punzo F. and Jellies J. (1983) Comparative water relations of araneid and lycosid spiderlings. Comp. Biochem. Physiol. 74A, 981-985. Punzo F. and Mutchmor J. A. (1980) Effects of temperature, relative humidity and period of exposure on the survival capacity of Tenebrio molitor (Coleoptera: Tenebrionidae). J. Kansas Enromol. Sot. 53, 26&270.
Riddle W. A. (1979) Metabolic compensation for temperature change in the scorpion Paruroctonus utahensis. J. Thermal Biol. 4, 125-128.
Riddle W. A. (1985) Hemolymph osmoregulation in several myriapods and arachnids. Comp. Biochem. Physiol. 80A, 313-323. Robertson H. G., Nicolson S. W. and Louw G. N. (1982) Osmoregulation and temperature effects on water loss and oxygen consumption in two species of African scorpion. Comp. Biochem. Physiol. 71A, 605609. Sokal R. R. and Rohlf F. J. (1981) Biometry: The Principles and Practice of Statistics in Biological Research, 2nd ed. W. H. Freeman & Co., New York. Warburg M. R. (1986) The distribution of water in the body compartments of four scorpion species. Comp. Biochem. Physiol. 84A, 637641. Warburg M. R. and Ben-Horin A. (1981) The response to temperature gradients of scorpions from mesic and xeric habitats. Comp. Biochem. Physiol. 68A, 277-279. Warburg M. R., Goldenberg S. and Ben-Horin A. (1980) Thermal effect on evaporative water loss and hemolymph osmolarity in scorpions at low and high humidities. Comp. Biochem. Physiol. 67A, 47-57.
Winston P. W. and Bates D. H. (1960) Saturated salt solutions for the control of relative humidity in biological research. Ecology 41, 232-237.
03~-9629/91 $3.00 + 0.00 Pergamon Press pit
Printed in Great Britain
THE EFFECTS OF TEMPERATURE AND MOISTURE ON SURVIVAL CAPACITY, CUTICULAR PERMEABILITY, HEMOLYMPH OSMOREGULATION AND METABOLISM IN THE SCORPION, CEiW’RUROIDES HENTZI (BANKS) (SCORPIONES, BUTHIDAE) FRED FUNZO Department
of Biology, University of Tampa. Tampa, FL 33606, U.S.A. Telephone: (813) 253-6206 (Received 25 March 1991)
Abstract-1. The temperature and water relations of Centruroides hentzi females were investigated. At I2 and 72% relative humidity (RH), the lower and upper LT% were -4.5 and 43.7”C, and -4.7 and 45.l”C, respectively. When exposed to high temperature stress, survivorship was significantly greater under mesic conditions. 2. Cuticular water loss was higher under xeric conditions (12% RH), ranging from 0.061 mg/cm2/hr at 30°C to 0.211 at 41°C. 3. Exposure to dry air (O-5% RH) resulted in a significant increase in hemolymph osmolality: from 441 to 688 mOsm over a 5 day period. 4. Mean oxygen consumption rates increased from 161.7 mm3/g/hr at 34°C to 541.6 at 44°C. ATPase activity was significantly higher in animals acclimated and tested at 35°C.
INTRODUCTION
The distribution, activity and survivorship of terrestrial anthropods are closely associated with their ability to withstand temperature and humidity stress while resisting evaporative water loss (EWL) (Edney, 1977; Punzo, 1989a,b). Although insects have received a great deal of attention (see reviews by Edney, 1977; Cloudsley-Thompson, 1973, there is significantly less information concerning the temperature and water relations of arachnids (Pulz, 1987). Most of the available arachnid literature concerns desert spiders and scorpions (Hadley, 1970, 1974; Warburg et al., 1980; Robertson et al., 1982; Pulz, 1987; Punzo, in press). Only a few studies have concerned themselves with the effects of temperature stress on mesic scorpions (Hadley, 1990). There is currently no data available on the thermal biology of the scorpion, Certtruroides hentzi (Banks), a common buthid associated with mesic as well as the more xeric sand pine-scrub habitats of south-central Florida. Experiments were conducted in order to determine upper (ULTSO) and lower (LLT5,,) lethal temperatures, the combined effects of temperature humidity (RH) on survival capacity,
and
relative
cuticuiar permeability (measured as EWL), and osmoregulation, the effects of temperature on oxygen consumption rates, and the effects of thermal acclimation on ATPase activity. MATERIALS AND METHODS
Adult C. hen& were collected from Hillsborough and Orange Counties, Florida, during the spring and summer months of 1989 and 1990. Specimens were collected using pitfall traps. Since most of the animals collected during this sampling period were adult females (32-42 mm,
0.241-0.473 g), temperature experiments were confined to this sex and life cycle stage. Scorpions were returned to the laboratory and maintained at 21°C and 70% RH under a 12L: 12D photo~riod regime. They were fed on a diet of crickets and mealworm larvae and provided with water on a weekly basis. Food and water were removed 48 hr prior to testing. Test temperatures were obtained through the use of a Precision Model 816 incubator (Chicago, IL). Relative humidity values within the incubator were obtained through the use of silica gel or saturated salt solutions as described by Winston and Bates (1960): silica gel (O-S%), LiCl(12%), NaCl (73 i 2%). All experiments were conducted under conditions of total darkness (Punt0 and Mutchmor. 1980). Lethal temperature and survival capacity determinarions Values for LLT% and ULT, were obtained according to the procedure described by Punzo and Huff (1989). To summarize, 10 scorpions were placed individually in glass containers. Four replicates of 10 animals yielded data on a total of 40 subjects for each experimental condition (RH values of 12 and 72%). For LLTw determinations, scorpions were exposed to low temperatures ranging from - 1.O to - 8.O”C at O.l”C intervals for a period of 1 hr under each condition of RH. Following the exposure interval, scorpions were removed from the incubator and returned to normal rearing conditions. The number surviving after 24 hr was recorded and expressed as per cent survival. Similar procedures were employed for ULTsO determinations. Scorpions were exposed to test temperatures ranging from 39 to 48°C at O.l”C intervals for a period of 1 hr under mesic (72% RH) and xeric (12%) conditions. These data were used to determine lethal temperatures (Table 1) as well as to assess the combined effect of low (LTS) and high (HTS) temperature stress and RH on survival capacity (Table 2). Evaporative water loss (EWL)/cuticular
permeability
All experiments were conducted in a desiccation apparatus previously described by Punzo and Jellies (1983). Air was recycled at a flow rate of 3.8 l/min and RH values (12 and 72%) were obtained as described above. Test 833
834
FREDPUNZO
Table 1. Mean upper (ULT& and lower (LLT~)lethal temperatures for adult female Cenrruroides hentri under xeric (12% RH) and mesic (72% RH) conditions (N = 40). One hour period of exposure. Values in parentheses represent +SE Relative humidity (%)
Test
Lethal temperatures (“C)
12 72 P’
ULT
LLT
43.7 (0.13) 45.1 (0.021 O.bl ’
-4.5 (0.05) -4.7 (0.04) NS ’
*Level of significance computed using Student’s r-test (Sokal and Rohlf, 1981). temperatures ranging from 30 to 41’C (Table 3) were used throughout these experiments. Air was bubbled through 5OOml of the saturated salt solution and then passed through cotton traps before entering the desiccation chamber housing the animals. Scorpions were placed individually in glass containers (five animals per replicate,
N = 25 for each test temperature) provided with small holes for air circulation. Animals were weighed individually on a Metler Analytical Balance to the nearest 0.005 mg at 1 hr intervals over a 24 hr period. EWL (cuticular permeability) was expressed as water (weight) loss (mg/cm2/hr) as described by Hadley (1990). The surface area of individual scorpions was obtained using the relationship, S = kW2”, where S represents surface area (cm2), W the body weight (mg), and k a constant (Edney, 1977). To obtain a value for k, scorpions of known weight were cleared in KOH. Their exoskeletons were spread over a grid paper containing 0.25 mm squares and measured with a Unitron H60 dissecting microscope. The k value obtained for adult females was 0.194 cm*/mg + 0.05. Hemolymph osmolality determinations
Hemolymph samples were obtained from each scorpion according to the method described by Punzo (1983). An incision was made in the abdominal region of CO,-anesthetized animals and hemolymph samples were collected in heparinized 1.0 ~1 pipettes. These pipettes were sealed at both ends with vasoline and frozen at - 30°C for subsequent osmolality determinations. Hemolymph samples were centrifuged for 20 min at 1000 rpm. Analyses were determined for 30 different scorpions exposed to temperature of 35°C at @5% and 72% RH in order to assess the influence of desiccating conditions on osmoregulation in this species Table 2. The effects of low (LTS) and high (HTS) temperature stress on the per cent survival of Centruroides henrzi females under xeric (12% RH) and mesic (72% RH) conditions (N = 40). One hour exposure period Relative humidity (%) 12
72
LTS (“C)
Per cent survival (%)
-6.0 - 5.0 -4.0 - 3.0 -2.0
33 63 60 73
-6.0 -5.0 -40 -3.0 -2.0
38 60 60 70
0
0
Table 3. The effects of temperature and relative humidity (RH) on cuticular permeability in Cenrruroides henrzi females after a 24 hr period. Cuticular permeability expressed as mean water (weight) loss in malcm’lhr (N = 251. Values in oarentheses noresent f SE
HTS (“0
Per cent survival (%)
39 40 41 42 43 44 45
98 95 80 63 55 10 0
39 40 41 42 43 44 45 46 47 48
95 95 93 83 73 63 50 25 5 0
A Wilcoxon Signed Rank Test (Sokal and Rohlf, 1981) showed the effects of RH (12 vs 72%) on per cent survival to be significant only under conditions of HTS (Z = 3.77, P < 0.01).
Water loss (mg/cm*/hr) Relative humidity (%) 12% RH 72% RH
temperature (“C) 30 31 32 33 34 35 36 37 38 39
0.061 0.062 0.067 0.073 0.083 0.092 0.137 0.161 0.167 0.175 0.184 0.21 I
40
41
(0.012) (0.011) (0.013) (0.010) (0.015) (0.01 I) (0.014) (0.021) (0.018) (0.016) (0.023j (0.025)
0.060 (0.012)
0.061 0.061 0.062 0.066 0.077 0.079 0.091 0.104 0.113 0.121 0.127
(0.014) (0.01 I) (0.013) (0.012) (0.014) (0.01 I) (0.009) (0.015) (0.008) io.011 j (0.013)
An ANOVA (Sokal and Rohlf, 1981) showed a significant effect of RH on cuticular permeability (F = 2 I .04, P < 0.001).
(Table 4). Measurements were taken over a 5 day exposure period. Hemolymph osmolality was determined using a Knauer osmometer and expressed as mOsm/g. Oxygen consumption determination and the effects of thermal acclimation on ATPase activity Experiments were also conducted in order to assess the effects of thermal acclimation at the enzymic (molecular) level in order to more fully understand the effect of temperature on metabolic processes in this species. ATPase activity was chosen owing to its overall significance in locomotor activity in arthropods. Scorpions (N = 40) were acclimated at either 15 or 35°C (Table 5). Homogenates were prepared using cephalothoracic tissue from C. hentzi females. Tissues from five scorpions were pooled for each test. Tissues were homogenized in a Frey Model 70 tissue homogenizer at 0°C with deionized water. ATPase determinations were conducted according to the method described by Anderson and Mutchmor (1971) and recorded as pg P/mg/animal/min. Phosphorus determinations were obtained with a Coleman Model 24 spectrophotometer at 600 urn and compared to reaction blanks (Punzo and Huff, 1989). The effects of temperature on metabolic rate (oxygen consumption) was also determined and expressed as mm’/g/hr as described by Hadley and Hill (1969). Oxygen consumption was measured using a constant pressure respirometer as described by Engelmann (1963). Forty scorpions were placed individually inside the respirometer. The apparatus was then sealed with stopcock grease and submerged in a Tectronic water bath (Chicago, IL). Oxygen consumption was measured over a temperature range of 34 to 44°C at 2.O”C intervals (Table 6). Readings were recorded every 30 min over a 24 hr period. Scorpions were weighed Table 4. Hemolympb osmolality (mOsm/g i SE) in Centruroideshenlzi females exposed to a temperature of 35°C under xeric and mesic conditions (N = 30 for each test condition). Measurements were taken over a 5 day exposure period. Values expressed as means * SE
Exposure (davs) I
2 3 4 5
Hemolymph osmolality (mOsm/g + SE) Relative humidity 72% &5% 441*21 504 * 33 521 f 47 601 f 39 678+44
422 i I7 431 i 29 439 i 37 443&21 461 + 35
An ANOVA (Sokal and Rohlf, 1981) showed a signilicant effect of relative humidity (F = 34.17. P < 0.01) on hemolymph osmoregulation.
Temperature and water relations in the scorpion Table 5. ATPase activity hg P/mg/min) in cold (15°C) and warm (35°C) acclimated Cenfruroides henrzi females. Values expressed as means f 1.0 SD (N = 40 for each experimental condition) Acclimation temperature (“C) 15 15 35 35
Test temperature (“C) 15 35 35 15
‘Level of significance computed (Sokal and Roblf, 1981).
ATPase activity 0.367 0.396 0.892 0.514
+ f + f
0.03 0.02 0.1 I* 0.07
using Student’s f-test, P < 0.01
before each test and body volumes determined by water displacement (0.01 ml) after testing. The respirometer allowed for only minimal movement by test animals. RESULTS The upper and lower lethal temperatures for C. henrzi are shown in Table 1. This scorpion can withstand significantly higher ambient temperatures under mesic conditions (451°C). When exposed to dry air, the ULTso value dropped to 43.7”C. Relative humidity had no similar effect on LLTso values. Previous studies have shown that ULTS for scorpions can range from 45 to 48°C (Hadley, 1990), and for some species can even exceed 50°C for short exposure periods. With respect to survival at low temperatures, body temperatures between - 11.9 and - 5.8”C have been reported for some higher altitude species (Hadley, 1974, 1990). The combined effects of low and high temperature stress on survival capacity as a function of RH are shown in Table 2. Under conditions of LTS, RH had no significant effect on survival rates. When exposed to HTS (4144°C) however, survival capacity was higher under mesic conditions. In dry air, 90% of the scorpions died when exposed to 44°C. At this temperature mortality decreased to 37% under mesic conditions. This is in general agreement with results reported for other mesic scorpions (Warburg et al., 1980; Warburg and Ben-Hoi-in, 1981; Robertson et al., 1982; Hadley, 1990) and suggests that cooling via EWL is minimal in these species. This is in contrast to results reported for mesic and xeric insects which have demonstrated the ability to withstand higher temperatures under dry air conditions due to effective evaporative cooling mechanisms for short periods of time (CloudsleyThompson, 1975; Punzo and Mutchmor, 1980; Punzo and Huff, 1989; Punzo, 1989b, in press). The effects of temperature and RH on EWL (cuticular permeability) are shown in Table 3. Water loss rates were significantly higher under xeric Table 6. Relationahip between air temperature and mean oxygen consumption rates in Centruroides hentzi females (N = 40). Values in parentheses represent *SE Temperature (“C) 34 36 38 40 42 44
Mean oxygen consumption rate (mm’lglhr) 161.7 180.2 239.3 277.8 312.5 541.6
(14.4) (12.9) (21.6) (18.9) (17.8) (41.5)
on metabolic
conditions over the entire range of test temperatures. This, in conjunction with the results reported above for lethal temperatures and survival capacity, indicate that C. hentzi, normally associated with mesic habitats in Florida, is not particularly well adapted to dry air conditions. These water loss rates are in general agreement with those reported for other mesic and xeric scorpions such as Scorpio maurus (Scorpionidae), Nebo hierochonticus (Diplocentridae), Buthotus judaicus and Leiurus quinquestriatus (Buthidae) (Warburg et al., 1980) as well as Parabuthus villosus (Buthidae) and Opisthophthalmus cupensis (Scorpionidae) (Robertson et al., 1982). Thus, it would appear that many scorpions minimize EWL under xeric conditions through behavioral mechanisms such as fossoriality and nocturnal activity patterns (Hadley, 1990) which are characterized by more favorable microhabitat conditions. The results for hemolymph osmolality following a 5 day period of exposure to combinations of temperature and RH are shown in Table 4. Exposure to dry air resulted in an increase in hemolymph osmolality as compared to results obtained in moist air. Previous studies on several different species of scorpions have also reported an increase in hemolymph osmolality under experimental dehydration (Warburg et al., 1980; Robertson et al., 1982; Burton, 1984; Riddle, 1985). Similar results have been reported for insects (Edney, 1977; Punzo, 1989b) and spiders (Pulz, 1987) as well, although some species are osmoregulators rather than osmoconformers. The effects of thermal acclimation on ATPase activity are shown in Table 5. There was no significant effect of temperature in animals acclimated at 15°C and tested at 15 or 35°C. Only in animals acclimated at 35°C was a significant effect found. The increase in ATPase activity at higher temperatures indicates some capacity for molecular (enzymic) compensation to changing ambient temperatures in this species. Although this type of response is common in some insects (Anderson and Mutchmor, 1971; Punzo and Huff, 1989) it has not been generally associated with arachnids (Pulz, 1987; Punzo, in press). Changes in ATPase activity can influence locomotor activity and have obvious adaptive consequences for foraging, mating, habitat selection and escape responses. The relationship between air temperature and oxygen consumption rates (metabolism) is shown in Table 6. Increases in temperature resulted in a concomitant increase in metabolic rate. This is most pronounced at the 4244°C interval and agrees with similar studies on other terrestrial arthropods which have shown a more significant increase in oxygen consumption rates as upper lethal temperatures are approached (Hadley and Hill, 1969; CloudsleyThompson, 1975; Pulz, 1987). DISCUSSION
An ANOVA (Sokal and Rohlf, 1981)showed a significant effect of temperature (F = 41.04, P < 0.001).
835
rate
Centruroides hentzi is commonly associated with mesic habitats in Florida characterized by sandy substrates. It can also be found in less common habitats such as sand pine and scrub associations where more xeric conditions prevail. This scorpion can reduce its exposure to unfavorable environmental
836
FREDPUNZO
conditions through burrowing and nocturnal activity patterns which represent behavioral mechanisms of thermoregulation commonly associated with scorpions (Cloudsley-Thompson, 1975; Edney, 1977). Although these adaptive responses are especially evident in desert-dwelling species, they are rather common in mesic species as well (Hadley, 1990). The upper lethal temperatures for C. hentzi females (Table 1) are similar to those reported for other mesic and xeric scorpions (Warburg et al., 1980; Warburg and Ben-Horin, 1981; Robertson et al., 1982) and tend to be somewhat higher than those reported for other arachnids and insects found in similar habitats (Punzo and Jellies, 1983; Punzo, 1989b, 1991; Hadley, 1990). It is important to keep in mind that seasonal acclimatization effects have been reported to alter lethal temperatures in some scorpions (Riddle, 1985) and other arthropods (Cloudsley-Thompson, 1975; Punzo and Mutchmor, 1980; Punzo and Huff, 1989). It would be interesting to determine lethal temperatures for C. hentzi collected during different months of the year (fall and winter) and compare them to the values presented in this study for animals collected during spring and summer months. The data on survivorship as a function of temperature and RH stress (Table 2) indicate that C. henlzi females are more resistant to HTS under mesic conditions. This is supported by the preference for nocturnal activity exhibited by this species (personal observation) as well as its biogegraphical distribution and microhabitat preferences. The survival of terrestrial arthropods is predicated upon their ability to limit desiccation through EWL. Transpiratory water loss (cuticular and respiratory) is usually enhanced under the dry air conditions associated with xeric habitats. For this reason, many xeric arthropods are characterized by a desiccationand anatomical structures resistant epicuticle designed to close body cavities opening at the surface, as well as appropriate behavioral strategies 1974; Cloudsley-Thompson, 1975; (Hadley, Warburg, 1986; Pulz, 1987; Punzo, in press). EWL is also facilitated by the relatively small body size and concomitant large surface area to volume ratio associated with arthropods. As a group, scorpions possess an integument highly impermeable to water (Hadley, 1990) that is most highly developed in xeric species. Cuticular permeability (water loss rates) in C. hentzi (Table 3) is significantly higher when animals are exposed to dry air conditions. Previous studies have shown that water loss rates in other scorpions exposed to an air temperature of 30°C can range from 0.025 to 0.049 mg/cm*/hr for xeric species, and from 0.064 to 0.270 mg/cm2/hr for species from more mesic habitats (Warburg et al., 1980; Robertson et al., 1982; Hadley, 1990). In C. hentzi, water loss rates at 30°C were 0.061 and O.O60mg/cm*/hr under xeric and mesic conditions, respectively. This is in agreement with the range of values mentioned previously for mesic scorpions. Previous investigators have also shown that over the temperature range used in this study, most of the EWL can be attributed to cuticular loss rather than respiration or excretion (Warburg et al., 1980). Where collecting conditions permit, future
studies should be conducted to analyse the relationship between temperature, RH and EWL rates for various life cycle stages (body size) for both sexes of C. hentzi. High temperature stress combined with exposure to dry air can lead to pronounced transitory water deficits in terrestrial arthropods regardless of behavioral and physiological mechanisms for reducing water loss. Such water loss can be accompanied by marked changes in hemolymph volume and osmolality (Riddle, 1985). Many insects have shown the ability to regulate the osmotic pressure of hemolymph despite the reduction of hemolymph volume normally associated with dehydration (Edney, 1977) whereas most scorpions have demonstrated the capacity to tolerate increasing hemolymph osmolality associated with water loss (Hadley, 1970, 1990). Warburg et al. (1980) reported a significant increase in hemolymph osmolality for several scorpions exposed to dry air conditions over a 7 day period. Furthermore, hemolymph osmolality increased to a greater extent in xeric species. In the present study, there was a significant increase in hemolymph osmolality when C. hentzi females were exposed to desiccating conditions (Table 4). At O--5% RH, individuals of this species were able to withstand an increase from 441 to 678 mOsm/g indicating that this species, like most scorpions, is an osmoconformer. One exception to this was reported by Robertson et al. (1982) for Parabuthus villosus, a large, non-burrowing scorpion inhabiting very arid regions of Ethiopia and frequently active during the day. This species exhibited the capacity for efficient ionic and osmotic regulation that compared with that demonstrated for some desert insects. More information is needed to assess the mechanisms responsible for osmoregulation in P. viflosus. The metabolic rates determined for C. hentzi (Table 6) are comparable to those reported for other scorpions (Hadley and Hill, 1969; Robertson et al., 1982), and are generally lower than values reported for insects (Punzo, 1989b). ATPase activity showed an acclimatory response at 35°C (Table 5). Presumably, this indicates a relationship between change in temperature and locomotor activity at the enzymic level associated with enzyme-substrate affinity allowing for maximal movement over a range of ambient temperatures. This will require further investigation in light of the fact that previous studies have reported the absence of a thermal acclimatory response for oxygen consumption rates in scorpions (Robertson et al., 1982) and other arachnids (Pulz, 1987; Punzo, 1991). This has been attributed to the markedly lower metabolic rates exhibited by spiders and scorpions. However, Riddle (1979) reported that high temperature acclimation results in a lateral shift in the metabolic ratetemperature curve in the desert scorpion, Paruroctonus utahensis. Acknowledgemenfs-I wish to thank Mr T. Punzo who assisted in the collection of specimens, and Dr Brian Garman, Department of Mathematics, who provided valuable assistance in the statistical analyses. A Faculty Development Grant from the University of Tampa made much of this work possible.
Temperature and water relations in the scorpion REFERENCES Anderson R. L. and Mutchmor J. A. (1971) Temperature acclimation in Tribolium and Musca at locomotory, metabolic and enzymic levels. J. Insect Physiol. 17, 22052219. Burton R. F. (1984) Hemolymph composition in spiders and scorpions. Comp. Biochem. Physiol. 78A, 613-616. Cloudsley-Thompson J. L. (1975) Adaptations of Arthropoda to arid environments. A. Rev. Entomol. 20, 261-283. Edney E. B. (1977) Water Balance in Land Arthropods. Springer, Berlin. Engelmann M. D. (1963) A constant pressure respirometer for small arthropods. Entomol. News 74, 181-187. Hadley N. F. (1970) Water relations of the desert scorpion, Hadrurus arizonensis. J. exp. Biol. 53, 547-558.
Hadley N. F. (1974) Adaptational biology of desert scorpions. J. Arachnol. 2, 11-23. Hadley N. F. (1990) Environmental physiology. In The Biology of Scorpions (Edited by Polis G. A.), pp. 321-340. Stanford University Press, Stanford, California. Hadley N. F. and Hill R. D. (1969) Oxygen consumption of the scorpion Centruroides sculpturatus. Comp. Biochem. Physiol. -29, 217-226.
_
Pulz R. (1987) Thermal and water relations. In Ecoohvsiology oj Spiders (Edited by Nentwig W.), pp.’ 2655. Springer, New York. Punzo F. (1983) Hemolymph chemistry of the spiders, Heteropoda venatoria (Sparassidae), Pisaurina mira (Pisauridae) and Amaurobius bennerti (Amaurobiidae). Comp. Biochem. Physiol. 75A, 647652.
Punzo F. (1989a) Composition of the hemolymph of mygalomorph spiders (Orthognatha). Comp. Biochem. Physiol. 93A, 757-760.
Punzo F. (1989b) Comparative temperature and water relations and hemolymph osmoregulation in the desert insects, Taeniopoda eques and- Schistocerca vaga (Orthoptera, Acrididae). Comp. Biochem. Physiol. 93A, 751-755.
837
Punzo F. Intraspecific variation to thermal stress in the tarantula, Dugesiellu echina Chamberlin (Orthognatha, Theraphosidae). Bull. Br. Arachnol. Sot. (in press). Punzo F. and Huff G. (1989) Comparative temperature and water relations and the effects of thermal acclimation on Tenebrio molitor and Tenebrio obscurus (Coleoptera: Tenebrionidae). Comp. Biochem. Physiol. 93A, 527-533. Punzo F. and Jellies J. (1983) Comparative water relations of araneid and lycosid spiderlings. Comp. Biochem. Physiol. 74A, 981-985. Punzo F. and Mutchmor J. A. (1980) Effects of temperature, relative humidity and period of exposure on the survival capacity of Tenebrio molitor (Coleoptera: Tenebrionidae). J. Kansas Enromol. Sot. 53, 26&270.
Riddle W. A. (1979) Metabolic compensation for temperature change in the scorpion Paruroctonus utahensis. J. Thermal Biol. 4, 125-128.
Riddle W. A. (1985) Hemolymph osmoregulation in several myriapods and arachnids. Comp. Biochem. Physiol. 80A, 313-323. Robertson H. G., Nicolson S. W. and Louw G. N. (1982) Osmoregulation and temperature effects on water loss and oxygen consumption in two species of African scorpion. Comp. Biochem. Physiol. 71A, 605609. Sokal R. R. and Rohlf F. J. (1981) Biometry: The Principles and Practice of Statistics in Biological Research, 2nd ed. W. H. Freeman & Co., New York. Warburg M. R. (1986) The distribution of water in the body compartments of four scorpion species. Comp. Biochem. Physiol. 84A, 637641. Warburg M. R. and Ben-Horin A. (1981) The response to temperature gradients of scorpions from mesic and xeric habitats. Comp. Biochem. Physiol. 68A, 277-279. Warburg M. R., Goldenberg S. and Ben-Horin A. (1980) Thermal effect on evaporative water loss and hemolymph osmolarity in scorpions at low and high humidities. Comp. Biochem. Physiol. 67A, 47-57.
Winston P. W. and Bates D. H. (1960) Saturated salt solutions for the control of relative humidity in biological research. Ecology 41, 232-237.