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sorbitol contents of the house spider, Achaearanea tepidariorum (Araneae: Theridiidae)
C‘on~p. Eiochem. Pergamon
Phwiol. Vol. I IOB, No. 3. pp. 539-545, 1995 Copyright Q 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491195 $9.50 + 0.00
Seasonal change in glycogen and inositol/sorbitol contents of the house spider, Achaearanea tepidariorum (Araneae: Theridiidae) Kazuhiro Tanaka Zoological Japan
Section,
Institute
of Low Temperature
Science,
Hokkaido
University,
Sapporo
060,
The house spider, Achaearanea tepidariorum, accumulated three kinds of polyols, namely scyllo-inositol, myo-inositol and sorbitol during hibernation. In natural population, production of scyllo-inositol and myo-inositol began earlier and the pool was maintained longer than sorbitol. Accumulation of the two isomers of inositol began with the induction of diapause; photoperiod was a primary factor triggering the synthesis, and temperature had secondary influence. Sorbitol synthesis required not only short-day induced diapause state, but also exposure to low temperature. Glycogen was a possible precursor of sorbitol. The seasonal profiles of these carbohydrate changes were reconstructed under artificial photoperiodic and thermal conditions. The triggering mechanism and the importance of polyol synthesis in overwintering are discussed. Key words: Diapause; Sorbitol; Temperature. Comp. Biochem.
Physiol.
Glycogen;
Myo-inositol;
llOB, 539-545,
Overwintering;
Photoperiod;
Scyllo-inositol;
1995.
Introduction reported (e.g. Miller and Smith, 1975; Gehrken, 1984; Shimada et al., 1984; Hamilton et al., 1985; Storey and Storey, 1986; Hoshikawa, 1987). In general, fat body glycogen is a major source of these metabolites (Storey and Storey, 1991). The synthesis of polyols and sugars is often triggered by low temperature (Hayakawa and Chino, 1981; Baust, 1982; Storey and Storey, 1983) but, in some insects, it is closely related to dispause state (Chino, 1957; Lee et al., 1987; Pullin and Bale, 1989). Surveys of cryoprotectants has mainly been made among insects, there are few studies in spiders. The araneid, Araneus cornutus, accumulates glycerol during the hibernation (Kirchner and Kestler, 1969). Glycerol is also detected in the hibernating immature of Philodromis and Cfubiona species, together with a thermal hystersis proteins (Duman, 1979). On the other hand, no polyol or sugar has been detected in the hibernating specimens of the garden spider, Argiope auruntiu (Riddle, 1981) and the desert
Many arthropods accumulate low molecular weight polyols and/or sugars as significant metabolites during hibernation (for reviews see Somme, 1982; Storey and Storey, 1991). These are assumed to have function as cryoprotectants or fuel for basal metabolism during winter, or as simple responses to metabolic suppression (Lee, 1991, Pullin et al., 1991; Storey and Storey, 1991; Pullin, 1992). Glycerol is a common polyol accumulated in winter (see Asahina, 1969 and Somme, 1982 for lists of cryoprotectants produced by insects, mites and spiders), but other polyols such as sorbitol, myo-inositol, threitol, ribitol and ethylene glycol, or sugars such as glucose, fructose and trehalose are also
to: K. Tanaka, Zoological Section, Institute of LOW Temperature Science, Hokkaido University, Sapporo 060, Japan. Tel. (011) 716-211 I; Fax (01 I) 706-7 142. Received 18 May 1994; revised 18 August 1994; accepted 24 August 1994. Correspondence
539
540
K. Tanaka
spider, Agelenopsis aperta (Lee and Baust, 1985). The overwintering carbohydrate metabolism in spiders is thus diverse among species. The purpose of this study is to characterize the winter carbohydrate metabolism of another spider, Achaearanea tepidariorum. In northern Japan, this spider passes the winter at both the nymphal and imaginal stages in a state of diapause (Tanaka, 1989, 1991, 1992). In the present study, the multiple polyol system of this spider and the seasonal change of glycogen and polyol contents in the natural population are reported. In addition, the influence of photoperiod, temperature and diapause state on the regulation of glycogen and polyol levels are also analysed.
Materials and Methods Monthly
samples
of
the
house
spider,
Achaearanea tepidariorum were collected from
the campus of Hokkaido University, Sapporo (43”03’N) immediately before the glycogen and polyol determination. In this locality, various sizes of nymphs and adult were found throughout the year (K. Tanaka, unpublished observations), only nymphs of more than 5 mg wet weight were used in this study. Laboratory cultures were established from a female collected on the campus of Hokkaido University in July 1992 and maintained at 20°C under diapause avoiding photoperiod (16 hr light : 8 hr dark or LD 16 : 8). Approximately 90 spiderlings were obtained from a egg sac produced by the mother spider. Individual spiderlings were reared separately in a glass tube as described by Tanaka (1991). To elucidate the influence of photoperiod and temperature on the carbohydrate metabolism, the hatchlings were divided into four groups and were kept at either a diapause avoiding (LD 16:8) or diapause inducing photoperiod (LD 12: 12) with two different temperature regimes (20 and 17°C). To elucidate the influence of low temperature on polyol production, spiders were put individually into a gelatine capsule and exposed to various temperatures using a deep freezer (Ebara, Tokyo, ESL-70 A) equipped with a fan heater (Hitachi, Tokyo, VF-1011) and a programmable heater controller (Omuron, Osaka, E5T-R9 1P). Low molecular weight carbohydrates such as sugars and polyols were detected by gas chromatography. According to Shimada et al. (1984) samples were homogenized individually with 4 ml of 80% ethanol in a glass homogenizer and then 1 mg erythritol was added as an internal standard. The homogenate was centrifuged at 3000g for 15 min and the super-
natant evaporated in a vacuum at 50°C until dryness. To the residue, 0.05 ml trimethylsilylating reagent (TMSI-C, GL Sciences Inc., Tokyo) was added and the solution was heated at 65°C for 40 min. The resulting derivative was injected to a gas chromatograph (Shimazu, Kyoto, GC4CMPF) with a glass column (3 mm x 3 m) containing 1.5% OV-1. The column was heated from 130 to 270°C at 5°C min and then kept at that temperature for 10 min. To determine glycogen content, 2 ml of 10% trichloroacetic acid was added the sediment of the homogenate. The mixture was boiled at 100°C for 15 min and then centrifuged at 3000 g for 10min. The glycogen content in the supernatant was determined by the anthrone/ sulphuric acid method (Trevelyan and Harrison 1952).
Results IdentiJication of carbohydrates
A chromatographic profile of the extract from of the hibernating nymph is shown in Fig. 1. At least three major peaks were detected in the extracts. From the retention time, those were identified as scyllo-inositol, myo-inositol and sorbitol. In addition, glucose was sometimes detected, but the content was usually less than 0.1 mg/g wet weight. Seasonal changes in polyols and glycogen contents
Overwintering profiles of polyols and glycogen levels are shown in Fig. 2. For scyllo-inositol, a significant rise in the content was first observed in September. The content gradually
_____ 0
10
30
20 RETENTION
TIME
40
(min.)
Fig. I. A cromatographic profile of polyol derivatives extracted from hibernating nymph of Achaearanea tepidariown taken in January. The peaks are ERY: erythritol as an internal standard, GLU: glucose, SOR: sorbitol, S-INO: scyllo-inositol and M-INO: myo-inositol.
Winter carbohydrate metabolism in spider
541
newly hatched spiders were reared under either short-day (LD 12:12) or long-day (LD 16:8) conditions with different temperature regimes (200 and 17°C) and the changes in glycogen and polyol contents were monitored. At both 20” and 17°C photoperiod had great influence on glycogen and inositol accumulation (Fig. 3). Individuals exposed to short-days accumulated significant amounts of glycogen, scylloinositol and myo-inositol (more than 40 mg/g in a \ glycogen and more than 1 mg/g in inositols), while those under the long-day conditions had only trace of scyllo-inositol and myo-inositol Sorbilol (less than 0.1 mg/g) and lower glycogen content (less than 16mg/g). & ._T .__ .i._e. _OL-_Rearing temperature also affected the inositol Scyllo-lnositol 81 content. For both scyllo-inositol and myo-inositol, the contents tended to be higher in the lower temperature (17°C) than in the higher I/‘\_; y o-hosit 8’ temperature (20°C) (Mann-Whitney U-test, P < 0.05, days 70 and 90). On the other hand, j no difference was observed in the glycogen content between 20 and 17°C. Accumulation of glycogen and inositol were Fig. 2. Seasonal changes in gylcogen, scyllo-inositol, myoclosely related to the diapause state. Under the inositol and sorbitol contents of Achaearane fepiduriorum in natural population from August 1991 to June 1992 short-day conditions, diapause was induced (mean f SD) (N = 4-8). when spiders sum up the short-day cycles more than 40 days (Tanaka, 1991). Since the signifiincreased from 0.2 mg/g wet weight in Septem- cant rise of glycogen and inositol contents began ber to 4.5 mg/g in December. From December to occur at the day 40, which roughly correto March, the concentration was fairly stable at sponded with the required day numbers for around 4.5 mg/g. In April, the content began to diapause induction, it is concluded that both the decrease and finally disappeared in May. Myo- glycogen and inositol accumulation are cominositol concentration also began to increase in ponents of diapause syndrome. September and reached to the plateau in DeIt was noticeable that no sorbitol was accucember to January (average 9.6 mg/g). In mulated in the above experiments, even if diaFebruary, it began to decrease and finally disap- pause was induced. The independence of peared in May. sorbitol synthesis from short-day photoperiod Seasonal occurrence of sorbitol was more or diapause state suggested that there must be restricted to the winter season than the two another factor evoking its synthesis. According isomers of inositol. Sorbitol became detectable to the evidence that low temperature is a priin November and the concentration increased mary factor causing polyol or sugar synthesis in rapidly in December (average 6.5 mg/g). From certain insects (Baust, 1982) the influence of February onward, the content gradually de- low temperature on sorbitol synthesis was tested creased and finally disappeared in April. in the spiders. The 70-day-old diapausing Glycogen, the storage form of carbohydrates, nymphs reared under the short-day conditions also showed a remarkable seasonal variation in at 20°C were exposed to -2°C for 6 days and the natural population. It increased rapidly in then the polyol contents were measured (Fig. 4). September and reached a maximum in NovemAfter cold acclimation, significant increase of ber (average 32.3 mg/g). From December, how- sorbitol and glucose were detected, thus low ever, the content decreased until March. The temperature must be a trigger of sorbitol and depletion in December was more or less con- glucose synthesis. In contrast, exposure of the comitant with a rapid increase of sorbitol con- 40-day-old non-diapausing nymphs reared tent, suggesting the conversion from glycogen to under the long-day conditions at 20” to -2°C sorbitol. for 2 days did not cause sorbitol synthesis (not shown). This means that diapause state may Efects of photoperiod and temperature on carbo also have influence on the sorbitol synthesis, but hydrate metabolism this effect can be seen only at low temperature. To elucidate the influence of photoperiod and Temperature dependent synthesis of sorbitol temperature on the carbohydrate metabolism, and glucose was also observed when the field
$-+--.
542
K. Tanaka LD 168
LD 12~12
sot Glycogen 40 tr 30
4.0
1
Myo-inositol
2.0 I 01
m--0-0.-c-.-.
0
30
60
Time after
0’
hatching (days)
Fig. 3. Changes in glycogen, scyllo-inositol and myo-inositol contents of Arhoeurunea repiduriorum under artificial photoperiod and temperature conditions. Open circle represents 20’C and closed circle 17’C. (N = 3)
collected spiders taken in October 1992 (average 11.6’C) were exposed to artificial temperature fall from 10 to -8’C at a rate of l”C/day (Table 1). As temperature was lowered, sorbitol was first detected at 0°C and finally reached to a mean of 10.1 mg/g wet weight at - 8°C. Glucose was first detected at 5°C and reached to 1.O mg/g at -8°C. Scyllo-inositol and myoinositol contents also increased as temperature decreases, but these had a peak at around 0°C. The thermal optima for synthesis were thus different among inositol and sorbitol. During the acclimation, glycogen content fluctuated. This might be due to in part the variability of glycogen content in the original field samples. On the other hand, from -4 to -PC, significant reduction in glycogen content occurred comcomitant with rapid increase of sorbitol content. This suggests that the preferential conversion from glycogen to sorbitol occurred at such low temperature.
Discussion Achaearanea tepidariorum accumulated three kinds of polyols (scyllo-inositol, myo-inositol and sorbitol) during hibernation. Among these polyols, occurence of scyllo-inositol is remarkable. This is the first report that a significant amount of this inositol is produced with dormancy, although the presence has been known from several insect species (Candy, 1967; Hipps et al., 1972). Myo-inositol is also rare substance in dormant arthropods and its increase in winter has been known from a few species (e.g. Bakken, 1985; Hoshikawa, 1987; Tanaka and Udagawa, 1993). Synthesis of scyllo-inositol and myo-inositol was a component of diapause syndrome; photoperiod was a primary trigger and temperature had secondary influence. On the other hand, the synthesis of sorbitol required not only entering diapause state but also low temperature. Somme
543
Winter carbohydrate metabolism in spider 4 i
12L 2ooc
2-
0i
3
--
-2°C Gdays
r
0 __ Glu
Sor
S-In0
i M-in0
Fig. 4. Influence of cold acclimation (- 2°C for 6 days) on polyol accumulation in 70 day-old diapausing nymphs (n = 3). Glu: Glucose, Sor: sorbitol, S-Ino: scyllo-inositol, M-Ino: myo-inositol.
(1982) classified the interaction between polyol/ sugar synthesis and diapause state into three categories as (1) non-diapausing species with temperature dependent synthesis, (2) diapausing species with temperature independent synthesis and (3) diapausing species with temperature dependent synthesis. According to this classification, the house spider may fall into the second category in terms of inositol synthesis, but into the third category in sorbitol synthesis. Under the field conditions, inositol and sorbitol showed different pattern in its seasonal variation (Fig. 2). Scyllo-inositol and myo-inositol begen to occur earlier and the pools were maintained longer than sorbitol pool. Such separate synthesis of two or more polyols have been also known in several insect species (Miller and Smith, 1975; Duman, 1980; Storey and Storey, 1986). In the golden rod gall fly, Eurosta solidaginis, for example, glycerol is first accumu-
lated in autumn and then sorbitol in winter. This is owing to both the temperature dependent enzyme regulation and differential trigger mechanisms; glycerol production is evoked by moderate warm temperature of lO-15°C while sorbitol production required cooler temperatures, below 3°C (Storey and Storey, 1983, 1986). Similar mechanism must be involved in the polyol production in the house spider. As mentioned above, thermal optima for inositol and sorbitol synthesis were different, so that we can reconstruct the separate polyol synthesis by their thermal responses. In the natural population, scyllo-inositol and myo-inositol contents began to increase in September (Fig. 2), when the diapause intervened in response to short-day conditions. The ambient temperature in September (average 18.4”C, minimum 8.9’C) were, however, too high to evoke sorbitol synthesis, so that the sorbitol synthesis would be inhibited until November when minimum temperature fall below 0°C (Table 2). Accumulation of low molecular weight carbohydrates has been correlated with an increase of insect cold hardiness (Lee, 1991). Previous study (Tanaka, 1993) reveals that chilling tolerance of this spider began to increase in September and reached a maximum level in January. This pattern more or less corresponds with the seasonal trends of scyllo-inositol and myo-inositol, thus suggesting their possible cryoprotective function. On the other hand, the seasonal occurence of sorbitol was late for the chilling tolerance, thus it appears that sorbitol has no effect at least in the early process enhancing the chilling tolerance. The function of sorbitol as cryoprotectant remains unknown. Recent studies offered another view on the relationship between polyol accumulation and cold tolerance; accumulation of polyol is simple responses to diapause mediated metabolic suppression and this metabolic suppression itself provides cryoprotection (Pullin et al., 1991; Pullin, 1992). This may also explain the observed correlation between the polyol contents and the level of chilling tolerance in this spider, although the experiments to test this possibility are yet to be carried out.
Table I. Effect of cold acclimation on carbohydrate contents (mean + SD mg/g wet weight) of the field collected spiders taken in October 1992 when ambient temperature averaged 1I .6’C Temperature (“C)
Glucose
Field 5 0 -4 -8
0.04 0.2 + 0.1 0.7 + 0.5 1.0*0.7
Sorbitol
S-Inositol
2.0+ 1.1 1.8 + 1.7 10.1 +4.1
1.8 + 0.5 2.3 + 0.3 2.8 + 0.6 2.1 +0.9 l.8f0.5
M-Inositol 2.4 + 2.7 + 6.0 + 4.1 + 4.7 f
1.3 0.5 1.2 1.7 1.3
Glycogen 32.1 + 17.3 + 23.7 + 18.7 + 4.7 +
5.1 2.6 3.9 6.2 2.8
Spiders were exposed to artificial temperature fall from 10°C to -8’C at a rate of l”C/day (N = 34).
544
K. Tanaka
In addition, low molecular weight carbohydrates are also considered to have a role as antifreeze agents which depress the supercooling point in a colligative manner. Supercooling point of this spider also has significant seasonal variation with a minimum in January (Tanaka, 1993) but this depression is directly caused by removal of gut ice nucleator rather than by increasing polyol concentration (K. Tanaka, unpublished observations). In most temperate insects studied, glycogen is usually converted into polyols or sugars in late autumn or early winter (e.g. Shimada et al., 1984; Storey and Storey, 1986; Hoshikawa, 1987; Rickards et al., 1987; Rickards and Shorthouse, 1989; Rojas et al., 1991). In the house spider, this is at least the case in the sorbitol synthesis. Reserve of glycogen was accumulated by the nymphs from September to November. In December and later, however, glycogen content gradually decreased with the concomitant rise of sorbitol content (Fig. 2). During cold acclimation experiments, particularly from - 4 to -8°C glycogen content decreased with increasing sorbitol (Table 1). Cold acclimation also caused glucose synthesis. This might be a rapid response to temperature fall and was observed mainly under laboratory conditions. During the cold acclimation, occurrence of glucose preceded the sorbitol synthesis, suggesting that it might be an intermediate product in the metabolic pathway from glycogen to sorbitol as demonstrated in several insect species (Storey and Storey, 1991). On the other hand, the relationship between glycogen and inositol was obscure. Under both natural and laboratory conditions, accumulation of scyllo-inositol and myo-inositol occurred concomitant with accumulation of glycogen storage, although the interconversion between glycogen and myo-inositol is demonstrated in the ladybeetle (Hoshikawa, 1987). The possible metabolic pathway of scyllo-inositol and myo-inositol in this spider was worthy of further investigation. Several insects reconvert polyol or sugar into glycogen in spring (e.g. Storey and Storey, 1986; Hoshikawa, 1987). In the house spider, however, the spring loss of sorbitol did not result in a resynthesis of glycogen; from December to March, glycogen content continued to decrease. This means that glycogen might be used at least a part as the fuel for basal metabolism during hibernation. Because of the responses to different environmental triggers, the winter carbohydrate metabolism of the house spider was thus highly complex. This is one of the characteristics of the overwintering biology of this spider. In the next step, we plan to determine the detail metabolic
Winter
carbohydrate
pathway of these polyols for further standing the overwintering adaptation.
under-
Acknowledgements--I would like to thank K. Shimada, Institute of Low Temperature Science, Hokkaido University for valuable suggestions during this work and for reading the original manuscript and T. Ohtsu, Faculty of Science, Hokkaido University for identification of polyols. This work was in part supported by JSPS Fellowships for Japanease Junior Scientists.
References Asahina E. (1969) Frost resistance in insects. Adu. Insect Physiol. 6, 149. Baust J. G. (1982) Environmental triggers to cold hardening. Comp. Biochem. Physiol. 13A, 563-570. Bakken H. (1985) Cold hardiness in the alpine beetles Pan&us septentriones and Calathus melanocephalus. J. Insect Physiol. 31, 447453. Candy D. J. (1967) Occurrence and metabolism of scylloinositol in the locust. Biochem. J. 103, 666-671. Chino Ii. (1957) Conversion of glycogen to sorbitol and glycerol in the diapausing egg of the Bombyx silkworm. Nature 180, 606-607. Duman J. G. (1979) Subzero temperature tolerance in spiders: The role of thermal hysteresis factors. J. camp. Physiol. 131, 341-352. Duman J. G. (1980) Factors involved in the overwintering survival of the freeze tolerant beetle, Dendroides canadensis. J. camp. Physiol. 136, 53-59. Gehrken U. (1984) Winer survival of an adult bark beetle Ips acuminatus Gyll. J. Insect Physiol. 30, 421429. Hamilton R. L., Mullins D. E. and Orcutt D. M. (1985) Freezing tolerance in the wood roach, Cryprocercus punctulatus. Experientia 41, 1535-1537. Hayakawa Y. and Chino H. (1981) Temperature-dependent interconversion between glycogen and trehalose in diapausing pupae of Philosamia Cynthia ricini and pryeri. Inset t Biochem. 11, 4347. Hipps P. P., William H. Holland H. and Sherman R (1972) Identification and measurement of chiro-inositol in the American cockroach, Periplaneta americana L. Biochem. biophys. Res. Commun. 46, 190331908. Hoshikawa K. (1987) Interconversion between glycogen and inositol in hibernating adults of a phytophagous ladybeetle, Epilachna vigintioctomaculata. Insect Biochem. 17, 265-,268. Kirchner W. and Kestler P. (1969) Untersuchungen zur kalteresistenz der schilfradspinne Araneus cornutus (Araneidae). J. Insect Physiol. 15, 41-53. Lee R. E. (1991) Principles of insect low temperature tolerance. In Insect at Low Temperature (Edited by Lee R. E. and Denlinger D. L.), pp. 17-46 Chapman & Hall, New York Lee R. E. and Baust J. G. (1985) Low temperature acclimation in the desert spider, Agelenopsis aperra (Araneae, Agelenidae). J. Arachnol. 13, 129-136. Lee R. E., Chen C. P., Meacham M. H. and Denlinger D. L. (1987) Ontogenetic patterns of coldhardiness and glycerol production in Sarcophaga crassipalpis. J. Insect Physiol. 33, 587-592. Miller L. K. and Smith J. S. (1975) Production of threitol and sorbitol by an adult insect: association with freezing tolerance. Nature 258, 519-520.
metabolism
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Pullin A. S. (1992) Diapause metabolism and changes in carbohydrates related to cryoprotection in Pieris brassicae. J. Insect Physiol. 38, 319-327. Pullin A. S. and Bale J. S. (1989) Influence of diapause and temperature on cryoprotectant synthesis and cold hardiness in pupae of Pieris brassicae. Comp. Biochem. Physiol. 94A, 499-503. Pullin A. S., Bale J. S. and Fontaine L. R. (1991) Physiological aspects of diapause and cold tolerance during overwintering in Pieris brassicae. Physiol. Entomol. 16, 447456. Riddle W. A. (1981) Cold survival of Argiope aurantia spiderlings (Araneae, Araneidae). J. Arachnol. 9,343-345. Rickards J. C. and Shorthouse J. D. (1989) Overwintering strategy of the stem-gall inducer Diplolepis spinosa (Hymenoptera: Cynipidae) in central Ontario. Can. J. Zool. 67, 2232-2237. Rickards J. C., Kelleher M. J. and Storey K. B. (1987) Strategies of freeze avoidance in larvae of the goldenrod gall moth, Epiblema scudderiana: winter profiles of a natural population. J. Insect Physiol. 33, 443450. Rojas R. R., Charlet L. D. and Leopord R. A. (1991) Biochemistry and physiology of overwintering in the mature larva of the red sunflower seed weevil, Smicronyx Jiil~us LeConte (Coleoptera: Curnuionidae). J. Insect Physiol. 31, 489496. Shimada K., Sakagami S. F., Honma K. and Tsutsui H. (1984) Seasonal changes of glycogen/trehalose contents, supercooling point and survival rate in mature larvae of the overwintering soybean pod borer, Leguminirora glycinivorella. J. Insect Physiol. 30, 369-373. Somme L. (1982) Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. 73A, 519-543. Storey K. B. and Storey J. M. (1991) Biochemistry of cryoprotectants. In Insects at Low Temperature (Edited by Lee R. E. and Denlinger D. L.), pp. 64-93, Chapman & Hall, New York. Storey, J. M. and Storey K. B. (1983) Regulation of cryoprotectant metabolism in the overwintering gall fly Eurosata solidaginis: temperature control of larva, glycerol and sorbitol levels. J. camp. Phwiol. 149, 495-502. Storey J. M. and Storey K. B. (1986) Winter survival of the gall fly larva, Eurosfa solidaginis: profiles of fuel reserves and cryoprotectants in a natural population. J. Insect Physiol. 32, 549-556. Tanaka K. (1989) Seasonal life cycle of the house spider. Achaearanea tepidariorum (Araneae, Theridiidae) in northern Japan. Appl. Entomol. Zool. 24, 117-125. Tanaka K. (1991) Diapause and seasonal life cycle strategy in the house spider, Achaearanea tepidariorum (Araneae, Theridiidae). Physiol. Entomol. 16, 249-262. Tanaka K. (1992) Photoperiodic control of diapause and climatic adaptation of the house spider, Achaearanea tepidariorum (Araneae, Theridiidae). Func. Ecol. 6, 5455552. Tanaka K. (1993) Seasonal change in cold tolerance of the house spider, Achaearanea tepidariorum (Araneae: Theridiidae). Acta arachnol. 42, 151-158. Tanaka K. and Udagawa T. (1993) Cold adaptation of the terrestrial isopod, Porcellio scaber to subnivean environments. J. camp. Physiol. 163B, 439444. Trevelyan W. E. and Harrison J. S. (1952) Studies on yeast metabolism. 1. Fractionation and microdetermination of cell carbohydrates. Biochem. J. 50, 2988303.
Phwiol. Vol. I IOB, No. 3. pp. 539-545, 1995 Copyright Q 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491195 $9.50 + 0.00
Seasonal change in glycogen and inositol/sorbitol contents of the house spider, Achaearanea tepidariorum (Araneae: Theridiidae) Kazuhiro Tanaka Zoological Japan
Section,
Institute
of Low Temperature
Science,
Hokkaido
University,
Sapporo
060,
The house spider, Achaearanea tepidariorum, accumulated three kinds of polyols, namely scyllo-inositol, myo-inositol and sorbitol during hibernation. In natural population, production of scyllo-inositol and myo-inositol began earlier and the pool was maintained longer than sorbitol. Accumulation of the two isomers of inositol began with the induction of diapause; photoperiod was a primary factor triggering the synthesis, and temperature had secondary influence. Sorbitol synthesis required not only short-day induced diapause state, but also exposure to low temperature. Glycogen was a possible precursor of sorbitol. The seasonal profiles of these carbohydrate changes were reconstructed under artificial photoperiodic and thermal conditions. The triggering mechanism and the importance of polyol synthesis in overwintering are discussed. Key words: Diapause; Sorbitol; Temperature. Comp. Biochem.
Physiol.
Glycogen;
Myo-inositol;
llOB, 539-545,
Overwintering;
Photoperiod;
Scyllo-inositol;
1995.
Introduction reported (e.g. Miller and Smith, 1975; Gehrken, 1984; Shimada et al., 1984; Hamilton et al., 1985; Storey and Storey, 1986; Hoshikawa, 1987). In general, fat body glycogen is a major source of these metabolites (Storey and Storey, 1991). The synthesis of polyols and sugars is often triggered by low temperature (Hayakawa and Chino, 1981; Baust, 1982; Storey and Storey, 1983) but, in some insects, it is closely related to dispause state (Chino, 1957; Lee et al., 1987; Pullin and Bale, 1989). Surveys of cryoprotectants has mainly been made among insects, there are few studies in spiders. The araneid, Araneus cornutus, accumulates glycerol during the hibernation (Kirchner and Kestler, 1969). Glycerol is also detected in the hibernating immature of Philodromis and Cfubiona species, together with a thermal hystersis proteins (Duman, 1979). On the other hand, no polyol or sugar has been detected in the hibernating specimens of the garden spider, Argiope auruntiu (Riddle, 1981) and the desert
Many arthropods accumulate low molecular weight polyols and/or sugars as significant metabolites during hibernation (for reviews see Somme, 1982; Storey and Storey, 1991). These are assumed to have function as cryoprotectants or fuel for basal metabolism during winter, or as simple responses to metabolic suppression (Lee, 1991, Pullin et al., 1991; Storey and Storey, 1991; Pullin, 1992). Glycerol is a common polyol accumulated in winter (see Asahina, 1969 and Somme, 1982 for lists of cryoprotectants produced by insects, mites and spiders), but other polyols such as sorbitol, myo-inositol, threitol, ribitol and ethylene glycol, or sugars such as glucose, fructose and trehalose are also
to: K. Tanaka, Zoological Section, Institute of LOW Temperature Science, Hokkaido University, Sapporo 060, Japan. Tel. (011) 716-211 I; Fax (01 I) 706-7 142. Received 18 May 1994; revised 18 August 1994; accepted 24 August 1994. Correspondence
539
540
K. Tanaka
spider, Agelenopsis aperta (Lee and Baust, 1985). The overwintering carbohydrate metabolism in spiders is thus diverse among species. The purpose of this study is to characterize the winter carbohydrate metabolism of another spider, Achaearanea tepidariorum. In northern Japan, this spider passes the winter at both the nymphal and imaginal stages in a state of diapause (Tanaka, 1989, 1991, 1992). In the present study, the multiple polyol system of this spider and the seasonal change of glycogen and polyol contents in the natural population are reported. In addition, the influence of photoperiod, temperature and diapause state on the regulation of glycogen and polyol levels are also analysed.
Materials and Methods Monthly
samples
of
the
house
spider,
Achaearanea tepidariorum were collected from
the campus of Hokkaido University, Sapporo (43”03’N) immediately before the glycogen and polyol determination. In this locality, various sizes of nymphs and adult were found throughout the year (K. Tanaka, unpublished observations), only nymphs of more than 5 mg wet weight were used in this study. Laboratory cultures were established from a female collected on the campus of Hokkaido University in July 1992 and maintained at 20°C under diapause avoiding photoperiod (16 hr light : 8 hr dark or LD 16 : 8). Approximately 90 spiderlings were obtained from a egg sac produced by the mother spider. Individual spiderlings were reared separately in a glass tube as described by Tanaka (1991). To elucidate the influence of photoperiod and temperature on the carbohydrate metabolism, the hatchlings were divided into four groups and were kept at either a diapause avoiding (LD 16:8) or diapause inducing photoperiod (LD 12: 12) with two different temperature regimes (20 and 17°C). To elucidate the influence of low temperature on polyol production, spiders were put individually into a gelatine capsule and exposed to various temperatures using a deep freezer (Ebara, Tokyo, ESL-70 A) equipped with a fan heater (Hitachi, Tokyo, VF-1011) and a programmable heater controller (Omuron, Osaka, E5T-R9 1P). Low molecular weight carbohydrates such as sugars and polyols were detected by gas chromatography. According to Shimada et al. (1984) samples were homogenized individually with 4 ml of 80% ethanol in a glass homogenizer and then 1 mg erythritol was added as an internal standard. The homogenate was centrifuged at 3000g for 15 min and the super-
natant evaporated in a vacuum at 50°C until dryness. To the residue, 0.05 ml trimethylsilylating reagent (TMSI-C, GL Sciences Inc., Tokyo) was added and the solution was heated at 65°C for 40 min. The resulting derivative was injected to a gas chromatograph (Shimazu, Kyoto, GC4CMPF) with a glass column (3 mm x 3 m) containing 1.5% OV-1. The column was heated from 130 to 270°C at 5°C min and then kept at that temperature for 10 min. To determine glycogen content, 2 ml of 10% trichloroacetic acid was added the sediment of the homogenate. The mixture was boiled at 100°C for 15 min and then centrifuged at 3000 g for 10min. The glycogen content in the supernatant was determined by the anthrone/ sulphuric acid method (Trevelyan and Harrison 1952).
Results IdentiJication of carbohydrates
A chromatographic profile of the extract from of the hibernating nymph is shown in Fig. 1. At least three major peaks were detected in the extracts. From the retention time, those were identified as scyllo-inositol, myo-inositol and sorbitol. In addition, glucose was sometimes detected, but the content was usually less than 0.1 mg/g wet weight. Seasonal changes in polyols and glycogen contents
Overwintering profiles of polyols and glycogen levels are shown in Fig. 2. For scyllo-inositol, a significant rise in the content was first observed in September. The content gradually
_____ 0
10
30
20 RETENTION
TIME
40
(min.)
Fig. I. A cromatographic profile of polyol derivatives extracted from hibernating nymph of Achaearanea tepidariown taken in January. The peaks are ERY: erythritol as an internal standard, GLU: glucose, SOR: sorbitol, S-INO: scyllo-inositol and M-INO: myo-inositol.
Winter carbohydrate metabolism in spider
541
newly hatched spiders were reared under either short-day (LD 12:12) or long-day (LD 16:8) conditions with different temperature regimes (200 and 17°C) and the changes in glycogen and polyol contents were monitored. At both 20” and 17°C photoperiod had great influence on glycogen and inositol accumulation (Fig. 3). Individuals exposed to short-days accumulated significant amounts of glycogen, scylloinositol and myo-inositol (more than 40 mg/g in a \ glycogen and more than 1 mg/g in inositols), while those under the long-day conditions had only trace of scyllo-inositol and myo-inositol Sorbilol (less than 0.1 mg/g) and lower glycogen content (less than 16mg/g). & ._T .__ .i._e. _OL-_Rearing temperature also affected the inositol Scyllo-lnositol 81 content. For both scyllo-inositol and myo-inositol, the contents tended to be higher in the lower temperature (17°C) than in the higher I/‘\_; y o-hosit 8’ temperature (20°C) (Mann-Whitney U-test, P < 0.05, days 70 and 90). On the other hand, j no difference was observed in the glycogen content between 20 and 17°C. Accumulation of glycogen and inositol were Fig. 2. Seasonal changes in gylcogen, scyllo-inositol, myoclosely related to the diapause state. Under the inositol and sorbitol contents of Achaearane fepiduriorum in natural population from August 1991 to June 1992 short-day conditions, diapause was induced (mean f SD) (N = 4-8). when spiders sum up the short-day cycles more than 40 days (Tanaka, 1991). Since the signifiincreased from 0.2 mg/g wet weight in Septem- cant rise of glycogen and inositol contents began ber to 4.5 mg/g in December. From December to occur at the day 40, which roughly correto March, the concentration was fairly stable at sponded with the required day numbers for around 4.5 mg/g. In April, the content began to diapause induction, it is concluded that both the decrease and finally disappeared in May. Myo- glycogen and inositol accumulation are cominositol concentration also began to increase in ponents of diapause syndrome. September and reached to the plateau in DeIt was noticeable that no sorbitol was accucember to January (average 9.6 mg/g). In mulated in the above experiments, even if diaFebruary, it began to decrease and finally disap- pause was induced. The independence of peared in May. sorbitol synthesis from short-day photoperiod Seasonal occurrence of sorbitol was more or diapause state suggested that there must be restricted to the winter season than the two another factor evoking its synthesis. According isomers of inositol. Sorbitol became detectable to the evidence that low temperature is a priin November and the concentration increased mary factor causing polyol or sugar synthesis in rapidly in December (average 6.5 mg/g). From certain insects (Baust, 1982) the influence of February onward, the content gradually de- low temperature on sorbitol synthesis was tested creased and finally disappeared in April. in the spiders. The 70-day-old diapausing Glycogen, the storage form of carbohydrates, nymphs reared under the short-day conditions also showed a remarkable seasonal variation in at 20°C were exposed to -2°C for 6 days and the natural population. It increased rapidly in then the polyol contents were measured (Fig. 4). September and reached a maximum in NovemAfter cold acclimation, significant increase of ber (average 32.3 mg/g). From December, how- sorbitol and glucose were detected, thus low ever, the content decreased until March. The temperature must be a trigger of sorbitol and depletion in December was more or less con- glucose synthesis. In contrast, exposure of the comitant with a rapid increase of sorbitol con- 40-day-old non-diapausing nymphs reared tent, suggesting the conversion from glycogen to under the long-day conditions at 20” to -2°C sorbitol. for 2 days did not cause sorbitol synthesis (not shown). This means that diapause state may Efects of photoperiod and temperature on carbo also have influence on the sorbitol synthesis, but hydrate metabolism this effect can be seen only at low temperature. To elucidate the influence of photoperiod and Temperature dependent synthesis of sorbitol temperature on the carbohydrate metabolism, and glucose was also observed when the field
$-+--.
542
K. Tanaka LD 168
LD 12~12
sot Glycogen 40 tr 30
4.0
1
Myo-inositol
2.0 I 01
m--0-0.-c-.-.
0
30
60
Time after
0’
hatching (days)
Fig. 3. Changes in glycogen, scyllo-inositol and myo-inositol contents of Arhoeurunea repiduriorum under artificial photoperiod and temperature conditions. Open circle represents 20’C and closed circle 17’C. (N = 3)
collected spiders taken in October 1992 (average 11.6’C) were exposed to artificial temperature fall from 10 to -8’C at a rate of l”C/day (Table 1). As temperature was lowered, sorbitol was first detected at 0°C and finally reached to a mean of 10.1 mg/g wet weight at - 8°C. Glucose was first detected at 5°C and reached to 1.O mg/g at -8°C. Scyllo-inositol and myoinositol contents also increased as temperature decreases, but these had a peak at around 0°C. The thermal optima for synthesis were thus different among inositol and sorbitol. During the acclimation, glycogen content fluctuated. This might be due to in part the variability of glycogen content in the original field samples. On the other hand, from -4 to -PC, significant reduction in glycogen content occurred comcomitant with rapid increase of sorbitol content. This suggests that the preferential conversion from glycogen to sorbitol occurred at such low temperature.
Discussion Achaearanea tepidariorum accumulated three kinds of polyols (scyllo-inositol, myo-inositol and sorbitol) during hibernation. Among these polyols, occurence of scyllo-inositol is remarkable. This is the first report that a significant amount of this inositol is produced with dormancy, although the presence has been known from several insect species (Candy, 1967; Hipps et al., 1972). Myo-inositol is also rare substance in dormant arthropods and its increase in winter has been known from a few species (e.g. Bakken, 1985; Hoshikawa, 1987; Tanaka and Udagawa, 1993). Synthesis of scyllo-inositol and myo-inositol was a component of diapause syndrome; photoperiod was a primary trigger and temperature had secondary influence. On the other hand, the synthesis of sorbitol required not only entering diapause state but also low temperature. Somme
543
Winter carbohydrate metabolism in spider 4 i
12L 2ooc
2-
0i
3
--
-2°C Gdays
r
0 __ Glu
Sor
S-In0
i M-in0
Fig. 4. Influence of cold acclimation (- 2°C for 6 days) on polyol accumulation in 70 day-old diapausing nymphs (n = 3). Glu: Glucose, Sor: sorbitol, S-Ino: scyllo-inositol, M-Ino: myo-inositol.
(1982) classified the interaction between polyol/ sugar synthesis and diapause state into three categories as (1) non-diapausing species with temperature dependent synthesis, (2) diapausing species with temperature independent synthesis and (3) diapausing species with temperature dependent synthesis. According to this classification, the house spider may fall into the second category in terms of inositol synthesis, but into the third category in sorbitol synthesis. Under the field conditions, inositol and sorbitol showed different pattern in its seasonal variation (Fig. 2). Scyllo-inositol and myo-inositol begen to occur earlier and the pools were maintained longer than sorbitol pool. Such separate synthesis of two or more polyols have been also known in several insect species (Miller and Smith, 1975; Duman, 1980; Storey and Storey, 1986). In the golden rod gall fly, Eurosta solidaginis, for example, glycerol is first accumu-
lated in autumn and then sorbitol in winter. This is owing to both the temperature dependent enzyme regulation and differential trigger mechanisms; glycerol production is evoked by moderate warm temperature of lO-15°C while sorbitol production required cooler temperatures, below 3°C (Storey and Storey, 1983, 1986). Similar mechanism must be involved in the polyol production in the house spider. As mentioned above, thermal optima for inositol and sorbitol synthesis were different, so that we can reconstruct the separate polyol synthesis by their thermal responses. In the natural population, scyllo-inositol and myo-inositol contents began to increase in September (Fig. 2), when the diapause intervened in response to short-day conditions. The ambient temperature in September (average 18.4”C, minimum 8.9’C) were, however, too high to evoke sorbitol synthesis, so that the sorbitol synthesis would be inhibited until November when minimum temperature fall below 0°C (Table 2). Accumulation of low molecular weight carbohydrates has been correlated with an increase of insect cold hardiness (Lee, 1991). Previous study (Tanaka, 1993) reveals that chilling tolerance of this spider began to increase in September and reached a maximum level in January. This pattern more or less corresponds with the seasonal trends of scyllo-inositol and myo-inositol, thus suggesting their possible cryoprotective function. On the other hand, the seasonal occurence of sorbitol was late for the chilling tolerance, thus it appears that sorbitol has no effect at least in the early process enhancing the chilling tolerance. The function of sorbitol as cryoprotectant remains unknown. Recent studies offered another view on the relationship between polyol accumulation and cold tolerance; accumulation of polyol is simple responses to diapause mediated metabolic suppression and this metabolic suppression itself provides cryoprotection (Pullin et al., 1991; Pullin, 1992). This may also explain the observed correlation between the polyol contents and the level of chilling tolerance in this spider, although the experiments to test this possibility are yet to be carried out.
Table I. Effect of cold acclimation on carbohydrate contents (mean + SD mg/g wet weight) of the field collected spiders taken in October 1992 when ambient temperature averaged 1I .6’C Temperature (“C)
Glucose
Field 5 0 -4 -8
0.04 0.2 + 0.1 0.7 + 0.5 1.0*0.7
Sorbitol
S-Inositol
2.0+ 1.1 1.8 + 1.7 10.1 +4.1
1.8 + 0.5 2.3 + 0.3 2.8 + 0.6 2.1 +0.9 l.8f0.5
M-Inositol 2.4 + 2.7 + 6.0 + 4.1 + 4.7 f
1.3 0.5 1.2 1.7 1.3
Glycogen 32.1 + 17.3 + 23.7 + 18.7 + 4.7 +
5.1 2.6 3.9 6.2 2.8
Spiders were exposed to artificial temperature fall from 10°C to -8’C at a rate of l”C/day (N = 34).
544
K. Tanaka
In addition, low molecular weight carbohydrates are also considered to have a role as antifreeze agents which depress the supercooling point in a colligative manner. Supercooling point of this spider also has significant seasonal variation with a minimum in January (Tanaka, 1993) but this depression is directly caused by removal of gut ice nucleator rather than by increasing polyol concentration (K. Tanaka, unpublished observations). In most temperate insects studied, glycogen is usually converted into polyols or sugars in late autumn or early winter (e.g. Shimada et al., 1984; Storey and Storey, 1986; Hoshikawa, 1987; Rickards et al., 1987; Rickards and Shorthouse, 1989; Rojas et al., 1991). In the house spider, this is at least the case in the sorbitol synthesis. Reserve of glycogen was accumulated by the nymphs from September to November. In December and later, however, glycogen content gradually decreased with the concomitant rise of sorbitol content (Fig. 2). During cold acclimation experiments, particularly from - 4 to -8°C glycogen content decreased with increasing sorbitol (Table 1). Cold acclimation also caused glucose synthesis. This might be a rapid response to temperature fall and was observed mainly under laboratory conditions. During the cold acclimation, occurrence of glucose preceded the sorbitol synthesis, suggesting that it might be an intermediate product in the metabolic pathway from glycogen to sorbitol as demonstrated in several insect species (Storey and Storey, 1991). On the other hand, the relationship between glycogen and inositol was obscure. Under both natural and laboratory conditions, accumulation of scyllo-inositol and myo-inositol occurred concomitant with accumulation of glycogen storage, although the interconversion between glycogen and myo-inositol is demonstrated in the ladybeetle (Hoshikawa, 1987). The possible metabolic pathway of scyllo-inositol and myo-inositol in this spider was worthy of further investigation. Several insects reconvert polyol or sugar into glycogen in spring (e.g. Storey and Storey, 1986; Hoshikawa, 1987). In the house spider, however, the spring loss of sorbitol did not result in a resynthesis of glycogen; from December to March, glycogen content continued to decrease. This means that glycogen might be used at least a part as the fuel for basal metabolism during hibernation. Because of the responses to different environmental triggers, the winter carbohydrate metabolism of the house spider was thus highly complex. This is one of the characteristics of the overwintering biology of this spider. In the next step, we plan to determine the detail metabolic
Winter
carbohydrate
pathway of these polyols for further standing the overwintering adaptation.
under-
Acknowledgements--I would like to thank K. Shimada, Institute of Low Temperature Science, Hokkaido University for valuable suggestions during this work and for reading the original manuscript and T. Ohtsu, Faculty of Science, Hokkaido University for identification of polyols. This work was in part supported by JSPS Fellowships for Japanease Junior Scientists.
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Pullin A. S. (1992) Diapause metabolism and changes in carbohydrates related to cryoprotection in Pieris brassicae. J. Insect Physiol. 38, 319-327. Pullin A. S. and Bale J. S. (1989) Influence of diapause and temperature on cryoprotectant synthesis and cold hardiness in pupae of Pieris brassicae. Comp. Biochem. Physiol. 94A, 499-503. Pullin A. S., Bale J. S. and Fontaine L. R. (1991) Physiological aspects of diapause and cold tolerance during overwintering in Pieris brassicae. Physiol. Entomol. 16, 447456. Riddle W. A. (1981) Cold survival of Argiope aurantia spiderlings (Araneae, Araneidae). J. Arachnol. 9,343-345. Rickards J. C. and Shorthouse J. D. (1989) Overwintering strategy of the stem-gall inducer Diplolepis spinosa (Hymenoptera: Cynipidae) in central Ontario. Can. J. Zool. 67, 2232-2237. Rickards J. C., Kelleher M. J. and Storey K. B. (1987) Strategies of freeze avoidance in larvae of the goldenrod gall moth, Epiblema scudderiana: winter profiles of a natural population. J. Insect Physiol. 33, 443450. Rojas R. R., Charlet L. D. and Leopord R. A. (1991) Biochemistry and physiology of overwintering in the mature larva of the red sunflower seed weevil, Smicronyx Jiil~us LeConte (Coleoptera: Curnuionidae). J. Insect Physiol. 31, 489496. Shimada K., Sakagami S. F., Honma K. and Tsutsui H. (1984) Seasonal changes of glycogen/trehalose contents, supercooling point and survival rate in mature larvae of the overwintering soybean pod borer, Leguminirora glycinivorella. J. Insect Physiol. 30, 369-373. Somme L. (1982) Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. 73A, 519-543. Storey K. B. and Storey J. M. (1991) Biochemistry of cryoprotectants. In Insects at Low Temperature (Edited by Lee R. E. and Denlinger D. L.), pp. 64-93, Chapman & Hall, New York. Storey, J. M. and Storey K. B. (1983) Regulation of cryoprotectant metabolism in the overwintering gall fly Eurosata solidaginis: temperature control of larva, glycerol and sorbitol levels. J. camp. Phwiol. 149, 495-502. Storey J. M. and Storey K. B. (1986) Winter survival of the gall fly larva, Eurosfa solidaginis: profiles of fuel reserves and cryoprotectants in a natural population. J. Insect Physiol. 32, 549-556. Tanaka K. (1989) Seasonal life cycle of the house spider. Achaearanea tepidariorum (Araneae, Theridiidae) in northern Japan. Appl. Entomol. Zool. 24, 117-125. Tanaka K. (1991) Diapause and seasonal life cycle strategy in the house spider, Achaearanea tepidariorum (Araneae, Theridiidae). Physiol. Entomol. 16, 249-262. Tanaka K. (1992) Photoperiodic control of diapause and climatic adaptation of the house spider, Achaearanea tepidariorum (Araneae, Theridiidae). Func. Ecol. 6, 5455552. Tanaka K. (1993) Seasonal change in cold tolerance of the house spider, Achaearanea tepidariorum (Araneae: Theridiidae). Acta arachnol. 42, 151-158. Tanaka K. and Udagawa T. (1993) Cold adaptation of the terrestrial isopod, Porcellio scaber to subnivean environments. J. camp. Physiol. 163B, 439444. Trevelyan W. E. and Harrison J. S. (1952) Studies on yeast metabolism. 1. Fractionation and microdetermination of cell carbohydrates. Biochem. J. 50, 2988303.