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Evolutionary Relationship between Diapause and Cold Hardiness in the House Spider, Achaearanea tepidariorum (Araneae: Theridiidae)
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PH: SOO22-191O(96)OOO91-1
J. InsectPhysiol.Vol. 43, No. 3, pp. 271–274, 1997 @ 1997Ekevier ScienceLtd All rights reserved.Printed in Great Britain 0022-1910/97$17.00 + 0.00
Evolutionary Relationship between Diapause and Cold Hardiness in the House Spider, Achaearanea tepidariorum (Araneae: Theridiidae) KAZUHIROTANAKA*T Received
23 April 1996; revised 2 July 1996
The relationshipbetween diapauseand cold hardinessof the house spider, Achaearaneatepidariorwn, differed geographically.In a cool-temperatepopulation, enhanced chilling tolerance
and supercoolingability were observed in diapause individuals,whereas a subtropicalpopulation showed only chilling tolerance.Becausethis spider is consideredto be of tropical origin, it would follow that the ancestraldiapauseof this spider was equippedwith chilling tolerance, but not with an increasedsupercoolingability. It seems that the ability to lower the supercooling point evolved through natural selection in the course of expansion of this species to the northern climates. @ 1997 Elsevier Science Ltd. All rights reserved Chillingtolerance Supercoolingpoint Diapause Geographicvariation
INTRODUCTION
Most insects and otherarthropodspecies overwintering in the cool-temperate and arctic regions enter diapause and develop cold-hardinessto cope with the severe winter climates (Danks, 1987;Tauber et al., 1986).Diapause and cold hardiness are both important ecophysiological traits for winter survival,but the relationshipbetween the two is not well understood (Denlinger, 1991). In the house spider, Achaearanea tepidariorum C Koch, the relationshipbetween diapause and cold hardiness differs geographically (Tanaka, 1996). Both cooltemperate and subtropicalpopulationsenter a facultative nymphaldiapausein responseto a short-dayphotoperiod (Tanaka, 1991, 1992),but depressionof the supercooling point (SCP) or temperature of ice crystallizationin the body is observed only in diapause-inducedindividuals of the cool-temperate population (Tanaka, 1996). This suggests that SCP depression is not always connected to diapause. The purpose of this study is to examine the relationship between diapause and cold hardiness of this spider
Achaearanea
tepidariorum
in more detail. The house spider is classified as a chill tolerant species by Bale’s definition(Bale, 1993),and is killed not only by freezing, but also by chilling without freezing (Tanaka, 1993).In the present study, I examine how closely the chilling tolerance and the supercooling ability are associated with diapause induction in two local populations of A. tepidariorum and discuss their evolutionaryrelationship. MATERIALS AND METHODS
Laboratory cultures of A. tepidariorum were established from femalescollectedon the campusof Hokkaido University, Sapporo (43”03’N) in May 1994 and on the campus of University of the Ryukyus, near Naha (26”12’N)in January 1994.The Sapporo populationwas regarded as a cool-temperate population and the Naha population as a subtropical population. Monthly mean and minimum temperature in the two study sites are given in Fig. 1. Spidersfor experimentswere obtainedeither from first or second egg-sacsdepositedby adults. Spiderlingswere reared individually in glass tubes (15 mm in diameter and 70 mm in height) and placed at either a diapause*ZoologicalSection, Institute of Low Temperature Science, Hokkaido preventing(16h light:8hdark or L:D 16:8)or a diapauseUniversity, Sapporo060, Japan. inducing photoperiod (L:D 12:12) at 20°C (Tanaka, tTo whom all correspondence should be addressed. Fax: 011-7061991, 1992). Spiders were fed Drosophila melanogaster 7142. 271
KAZUHIROTANAKA
introduced prey items (Drosophila melanogaster) were consideredas survivors.LLT 50 was calculatedby probit analysis (Torii et al., 1954). RESULTS
JFMAHJJASOND Monthly temperature at Sapporo (square) and Naha (triangle)(from ChronologicalScientificTables, 1996).Monthlymean temperature is represented by closed symbols and the average minimum temperature by open symbols.
adults for the first 30 days after hatching and then either field-collectedapterous Myrmica kotokui ants or Sepsis morrostigma adults every day. The photoperiods were controlledby a fluorescentlamp equipped with an automatic timer. The temperaturefluctuatedwithin *0.5°C of the desired level. Although both nymphs and adults of this spider have an ability to enter hibemal diapause (Tanaka, 1991, 1992), only nymphal spiders were used in the present study.Under the presentrearing conditions,the cool-temperate spidersentered diapausemainly at the 4th stadium and the subtropical spiders at the final (6th) stadium (Tanaka, 1991, 1992). A comparison of cold hardiness was therefore made between 4th-stadiumnymphs of the cool-temperate spiders (40-50 day-old non-diapausing nymphs and 70-80 day-old diapausingnymphs) and 6thstadium nymphs of the subtropical spiders (50-60 dayold non-diapausingnymphs and 80-90 day-old diapausing nymphs). Because the difference in stadia used may have some effect on the degree of cold hardiness, comparison of cold hardiness was only made within populations. According to previous studies (Tanaka, 1991, 1992), spiders staying at the same stadium longer than 30 days were regarded as diapause individuals. To determinethe SCP, each spiderwas put into a gelatine capsule in which the spiders were in contact with a tip of the thermocouple connecting to a recorder (Rikadenki,ICB681H).The capsule was held in a plastic vial (4.5 cm in diameter and 8 cm in height) for reducing the cooling rate to approximately 0.05°C/min in a freezer. SCP was determined by a release of the latent heat due to ice formation within the body. As an indicatorof chillingtolerance,the median lower lethal temperature(LLT 50) was determinedby exposing spiders to various temperatures ranging from – 1 to –9°C for 48h. After such exposure, they were returned to +18°C and were put into glass tubes (2 cm in diameter and 7 cm in height) individuallyto determine their survival 1 week later; only those spiders building a normal web and showing normal predatory behaviour against
A comparison of SCPS between diapause and nondiapausenymphs is given in Table 1. In the cool-temperate population,non-diapausespiders had a mean SCP of –7.0°C and diapause ones – 11.2°C. In the subtropical population,on the other hand, the SCP was –7 to –8°C and no significantdifferencewas foundbetween diapause and non-diapauseindividuals.These results agreed well to previous results (Tanaka, 1996). SCP depression was thus a component of the diapause syndrome only in the cool-temperatepopulation. Percentage survival of cool-temperateand subtropical spiders after 48h exposure to various subzero temperatures are shown in Fig. 2. It appears that many tested specimens died at a temperature much higher than their SCP. In the cool-temperatepopulation, for example, no diapause nymphs tolerated a temperature of –9”C, although their SCP was – 11°C on average (Table 1). Because their body had not been frozen, the cause of their mortality was probably due to chilling injury. In both the populations,the diapausenymphs survived better than the non-diapausecounterpartsat each subzero temperature, except for –1°C in the cool-temperate population(Fisher’sexact probabilitytest at 0.05’?/.level) (Fig. 2 and Table 1). These observations indicate that enhancementof chilling tolerance is associated with the diapause syndrome for both the populations. DISCUSSION The relationshipbetween diapause and cold hardiness of A tepidariorum is complicated and involves several interesting features. Firstly, this relationship changes depending upon which parameters of cold hardiness are used. In the subtropicalpopulation,the LLT 50 changed with diapause induction,but the SCP did not (Table 1).
TABLE1. Comparisonof SCPs (mean *SD) and LLT 50 for diapausing and non-diapausingnymphsof the cool-temperateand subtropical populationsof A. tepidariorum Climatic zone Cool-temperate Subtropical
Conditions’ ND D ND D
SCP(“c) –7.&ko.4 – 11.2*3.22 –7.2k0.7 –7.9+1.0
LLT 50 (“C) –1.9 –7.9 >– 1.03 –2.0
Sample size for SCP determinationare 21, 18, 19 and 25 from top to bottomand those for LLT 50 are reported in the caption for Fig. 2. ‘ ND, non-diapause;D, diapause. 2 Significantly lower than the non-diapausing individuals (Aspin– Welch method, t=5.42,p< O.05). 3LLT 50 could not be calculated,because of the relatively small number of survivors.
DIAPAUSEAND COLD HARDINESSIN SPIDER
100
o—
0 —0
—0
—
273
o \
(A)
o
●
\ \
Diapausing o
Non-diapausing
●
\ \
o \
“\e
0 -1
-2
-3
-4
-5
-6
-7
-8
o -9
Temperature c)
100o
T > .
z
.-> >
( (B)
50 “
\
0
Diapausing
9 u) \
o-
0,
0 \
● \N;n-dia*a”sing
r -1
-2
-3
-4
0 , -5
Temperature c) FIGURE2. Survivorshipof (A) the cool-temperateand (B) the subtropicalpopulationof A. tepidariorum after 48h expsure to various temperature regimes between – 1 and –9”C. Sample size ranged from 7 to 28 individualsper treatment.
This means that the underlying mechanisms regulating the supercoolingability and chilling tolerance are different from each other, as suggestedfor other freeze-avoiding arthropod species (e.g. Lee and Denlinger, 1985; Bennett and Lee, 1989; Pullin et al., 1991; Tanaka and Udagawa, 1993).It has been demonstratedthat the SCP depression of this spider is achieved partly by cessation of feeding activity and excretion or inactivation of the gut ice nucleators (Tanaka, 1994, 1996),while the LLT 50 depression is correlated with the concentration of scyllo-inositoland myo-inositol,possible cryoprotectants (Tanaka, 1995). Secondly, the relationshipbetween diapause and cold hardinesschangeswith locality.As summarizedin Table
2, diapause spiders of the cool-temperate spiders increased both chilling tolerance and supercooling ability,while those of the subtropicalspidersenhanced the chillingtoleranceonly. This may reflecta local difference in winter climates. In Sapporo,located in the cool-temperateregion, winter temperaturesfrequently fall below the level of the summer SCP and LLT 50 (Tanaka, 1993), so that reduction in SCP and LLT 50 prior to the winter is essential for winter survival of this spider. In Naha, located in the subtropicalregion, winter temperaturesoccasionallygo down to around +7”C, but no frost occurs (Tanaka, 1996).Under such mild winter conditions,the overwinteringspiders are normally never exposed to a subzero temperature, thus they would not
KAZUHIROTANAKA
274
TABLE2. Relationshipof diapause, chillingtolerance and supercooling ability in A. tepidariorum at different climatic zones Climatic zones
Diapause
Chilling tolerance
Supercooling ability
Cool-temperate Subtropical Tropical
+ + ?
+ + ?
+ — ?
+, Presence; –, absence.
need to lower the SCP for overwintering. Unlike the SCP, LLT 50 levels are lowered in diapause individuals of the subtropicalspiders(Table 1). At present,however, the adaptivesignificanceof this phenomenonis not clear. The present results demonstrated that non-diapause nymphs suffered a higher rate of mortality after chilling at –1°C than did their diapausecounterparts(Fig. 2), but winter temperatures in Naha never drop to such a low level. Afier 48h exposure to +7”C, which is the lowest temperatureever recorded in Naha, no mortalityoccurred in non-diapause nymphs of the Naha population (K. Tanaka, unpublished observations). Although tirther confirmationis necessary, it is possiblethat the observed increase in chilling tolerance in the subtropical population is a byproduct associated with the induction of diapause. Such a physiologicaltrait, however, may provide a mechanism which enables the subtropicalpopulation to explore more northern climatic regions. A. tepidariorum is considered to be of tropical origin (Levi, 1967) and the subtropicaldiapause is likely to be an ancestral type of the temperate diapause (Tanaka, 1992).If this scenario is correct, it would follow that the ancestral diapause of this spider evolved with chilling tolerance, but without an ability to lower the SCP. The latter ability would have been evolved secondarilyas the spiders expanded their distribution to the temperate regions(Tanaka, 1996).Spidersexpandingfrom the tropical to the temperate regions would first encounter a problem associated with chilling or non-freezing low temperaturerather than freezingtemperature.In the early phase of their movement to the north, therefore, natural selection may act to reduce winter mortality by improving chilling tolerance. Under such circumstances, the ability to increase chilling tolerance in association with diapause induction would gain an adaptive value, and would be reinforcedthroughnatural selection.As spiders expand tiwthernorth, they would require a mechanismto survive fhrther low temperatures,which would create an opportunity for them to evolve an ability to lower the
SCP. This study is the first to characterizea differentialgeographic profile in the relationshipbetween chilling tolerance and supercooling ability in relation to diapause induction. It has demonstratedthat the evolutionof cold
hardiness in A. tepidariorum involves at least several steps in association with diapause. How commonly a similar phenomenon is seen among other invertebrate species that have expanded their distribution from the tropical to the temperate regions is not known. It is necessary to examine many more species to evaluate the generality of the above phenomenon. REFERENCES Bale J. S. (1993) Classes of insect cold hardiness. Furrct Ecof. 7, 751-753. Bennett L. E. and Lee R. E. (1989) Simulatedwinter to summer transition in diapausing adults of the lady beetle (Hippodamia convergent): supercoolingpoint is not indicativeof cold hardiness. Physiol. Entomol. 14, 361-367.
Dsnks H. V. (1987) Insect dormancy: an ecological perspective. Biological surveyof Canada (terrestrial arthropods), Natural Museum of Natural Sciences, Ottawa. Denlinger D. L. (1991) Relationship between cold hardiness and diapause. In Insects at Low Temperature (Eds Lee Jr. R. E. and Denlinger D. L.), pp. 174198. Chapman and Hall, New York. Lee R. E. and Denlinger D. L. (1985) Cold tolerance in diapausing and non-diapausingstages of the flesh fly, Sarcophagi crassipalpis. Physiol. Entomol. 10,309-315. Levi H. W. (1967) Cosmopolitanand pantropical species of Theridiid spiders (Araneae: Theridiidae). Pac~@cInsects 9, 175–186. Pullin A. S., Bale J. S. and Fontaine X. L. R. (1991) Physiological aspects of diapause and cold tolerince during overwintering in Pieris brassicae. Physiol. Entom “ 16, 447456.
Tanaka K. (1991) Diapause and sef sonal life cycle strategy in the house spider, Achaearanea tepidariorum (Araneae, Theridiidae). Physiol. Entomol. 16, 249–262.
TanakaK. (1992)Photoperiodiccontrolof diapauseand climatic adaptation of the house spider, Achaearanea tepidariorum (Araneae, Theridiidse). Funct. ECOL6, 545-552. Tanaka K. (1993) Seasonal change in cold tolerance of the house spider, Achaearanea tepidariorum (Araneae: Theridiidae). Acta Arachnol. 42, 151-158.
Tanaka K. (1994) Effect of feeding and gut content on supercooling of the house spider, Achaearanea tepidariorum (Araneae: Theridiidae). C~o-Lett. 15, 361-366. Tanaka K. (1995) Seasonal change in giycogen and inositol/sorbitol contents of the house spider, Achaearanea tepidariorum (Arrmeae: Thendiidae). Comp. Biochem. Physiol. 11OB,539-545. Tanaka K. (1996) Seasonal and latitudinal variation in supercooling ability of the house spider, Achaearanea tepidariorum (Araneae: Theridiidae). Funct. Ecol. 10, 185-192. Tanaka K. and Udagawa T. (1993) Cold adaptation of the terrestrial isopod, Porcellio scaber, to subnivean environments. J. Comp. Physiol. B 163, 43944.
TauberM. J., TauberC. A. and Masaki S. (1986) Seasonal Adaptations in Insects. Oxford University Press, New York. Torii T., TakahashiK. and Doi L (1954) Statistical Methodsfor A4edical and Biological Research. Tokyo University Press, Tokyo. ,
Acknowledgements—I thank K. Shimada (Institute of Low Tempera-
ture Science, HokkaidoUniversity) and S. Tanaka (National Institute of Sericultural and Entomological Science) for reading the original manuscriptand H. Abe and Y. Saito (Facultyof Agriculture,Hokkaido University) for allowing me to use experimental facilities in the Laboratory of Applied Zoology. This research was in part supported by the JSPS Fellowshipfor Japanese Junior Scientists.
PH: SOO22-191O(96)OOO91-1
J. InsectPhysiol.Vol. 43, No. 3, pp. 271–274, 1997 @ 1997Ekevier ScienceLtd All rights reserved.Printed in Great Britain 0022-1910/97$17.00 + 0.00
Evolutionary Relationship between Diapause and Cold Hardiness in the House Spider, Achaearanea tepidariorum (Araneae: Theridiidae) KAZUHIROTANAKA*T Received
23 April 1996; revised 2 July 1996
The relationshipbetween diapauseand cold hardinessof the house spider, Achaearaneatepidariorwn, differed geographically.In a cool-temperatepopulation, enhanced chilling tolerance
and supercoolingability were observed in diapause individuals,whereas a subtropicalpopulation showed only chilling tolerance.Becausethis spider is consideredto be of tropical origin, it would follow that the ancestraldiapauseof this spider was equippedwith chilling tolerance, but not with an increasedsupercoolingability. It seems that the ability to lower the supercooling point evolved through natural selection in the course of expansion of this species to the northern climates. @ 1997 Elsevier Science Ltd. All rights reserved Chillingtolerance Supercoolingpoint Diapause Geographicvariation
INTRODUCTION
Most insects and otherarthropodspecies overwintering in the cool-temperate and arctic regions enter diapause and develop cold-hardinessto cope with the severe winter climates (Danks, 1987;Tauber et al., 1986).Diapause and cold hardiness are both important ecophysiological traits for winter survival,but the relationshipbetween the two is not well understood (Denlinger, 1991). In the house spider, Achaearanea tepidariorum C Koch, the relationshipbetween diapause and cold hardiness differs geographically (Tanaka, 1996). Both cooltemperate and subtropicalpopulationsenter a facultative nymphaldiapausein responseto a short-dayphotoperiod (Tanaka, 1991, 1992),but depressionof the supercooling point (SCP) or temperature of ice crystallizationin the body is observed only in diapause-inducedindividuals of the cool-temperate population (Tanaka, 1996). This suggests that SCP depression is not always connected to diapause. The purpose of this study is to examine the relationship between diapause and cold hardiness of this spider
Achaearanea
tepidariorum
in more detail. The house spider is classified as a chill tolerant species by Bale’s definition(Bale, 1993),and is killed not only by freezing, but also by chilling without freezing (Tanaka, 1993).In the present study, I examine how closely the chilling tolerance and the supercooling ability are associated with diapause induction in two local populations of A. tepidariorum and discuss their evolutionaryrelationship. MATERIALS AND METHODS
Laboratory cultures of A. tepidariorum were established from femalescollectedon the campusof Hokkaido University, Sapporo (43”03’N) in May 1994 and on the campus of University of the Ryukyus, near Naha (26”12’N)in January 1994.The Sapporo populationwas regarded as a cool-temperate population and the Naha population as a subtropical population. Monthly mean and minimum temperature in the two study sites are given in Fig. 1. Spidersfor experimentswere obtainedeither from first or second egg-sacsdepositedby adults. Spiderlingswere reared individually in glass tubes (15 mm in diameter and 70 mm in height) and placed at either a diapause*ZoologicalSection, Institute of Low Temperature Science, Hokkaido preventing(16h light:8hdark or L:D 16:8)or a diapauseUniversity, Sapporo060, Japan. inducing photoperiod (L:D 12:12) at 20°C (Tanaka, tTo whom all correspondence should be addressed. Fax: 011-7061991, 1992). Spiders were fed Drosophila melanogaster 7142. 271
KAZUHIROTANAKA
introduced prey items (Drosophila melanogaster) were consideredas survivors.LLT 50 was calculatedby probit analysis (Torii et al., 1954). RESULTS
JFMAHJJASOND Monthly temperature at Sapporo (square) and Naha (triangle)(from ChronologicalScientificTables, 1996).Monthlymean temperature is represented by closed symbols and the average minimum temperature by open symbols.
adults for the first 30 days after hatching and then either field-collectedapterous Myrmica kotokui ants or Sepsis morrostigma adults every day. The photoperiods were controlledby a fluorescentlamp equipped with an automatic timer. The temperaturefluctuatedwithin *0.5°C of the desired level. Although both nymphs and adults of this spider have an ability to enter hibemal diapause (Tanaka, 1991, 1992), only nymphal spiders were used in the present study.Under the presentrearing conditions,the cool-temperate spidersentered diapausemainly at the 4th stadium and the subtropical spiders at the final (6th) stadium (Tanaka, 1991, 1992). A comparison of cold hardiness was therefore made between 4th-stadiumnymphs of the cool-temperate spiders (40-50 day-old non-diapausing nymphs and 70-80 day-old diapausingnymphs) and 6thstadium nymphs of the subtropical spiders (50-60 dayold non-diapausingnymphs and 80-90 day-old diapausing nymphs). Because the difference in stadia used may have some effect on the degree of cold hardiness, comparison of cold hardiness was only made within populations. According to previous studies (Tanaka, 1991, 1992), spiders staying at the same stadium longer than 30 days were regarded as diapause individuals. To determinethe SCP, each spiderwas put into a gelatine capsule in which the spiders were in contact with a tip of the thermocouple connecting to a recorder (Rikadenki,ICB681H).The capsule was held in a plastic vial (4.5 cm in diameter and 8 cm in height) for reducing the cooling rate to approximately 0.05°C/min in a freezer. SCP was determined by a release of the latent heat due to ice formation within the body. As an indicatorof chillingtolerance,the median lower lethal temperature(LLT 50) was determinedby exposing spiders to various temperatures ranging from – 1 to –9°C for 48h. After such exposure, they were returned to +18°C and were put into glass tubes (2 cm in diameter and 7 cm in height) individuallyto determine their survival 1 week later; only those spiders building a normal web and showing normal predatory behaviour against
A comparison of SCPS between diapause and nondiapausenymphs is given in Table 1. In the cool-temperate population,non-diapausespiders had a mean SCP of –7.0°C and diapause ones – 11.2°C. In the subtropical population,on the other hand, the SCP was –7 to –8°C and no significantdifferencewas foundbetween diapause and non-diapauseindividuals.These results agreed well to previous results (Tanaka, 1996). SCP depression was thus a component of the diapause syndrome only in the cool-temperatepopulation. Percentage survival of cool-temperateand subtropical spiders after 48h exposure to various subzero temperatures are shown in Fig. 2. It appears that many tested specimens died at a temperature much higher than their SCP. In the cool-temperatepopulation, for example, no diapause nymphs tolerated a temperature of –9”C, although their SCP was – 11°C on average (Table 1). Because their body had not been frozen, the cause of their mortality was probably due to chilling injury. In both the populations,the diapausenymphs survived better than the non-diapausecounterpartsat each subzero temperature, except for –1°C in the cool-temperate population(Fisher’sexact probabilitytest at 0.05’?/.level) (Fig. 2 and Table 1). These observations indicate that enhancementof chilling tolerance is associated with the diapause syndrome for both the populations. DISCUSSION The relationshipbetween diapause and cold hardiness of A tepidariorum is complicated and involves several interesting features. Firstly, this relationship changes depending upon which parameters of cold hardiness are used. In the subtropicalpopulation,the LLT 50 changed with diapause induction,but the SCP did not (Table 1).
TABLE1. Comparisonof SCPs (mean *SD) and LLT 50 for diapausing and non-diapausingnymphsof the cool-temperateand subtropical populationsof A. tepidariorum Climatic zone Cool-temperate Subtropical
Conditions’ ND D ND D
SCP(“c) –7.&ko.4 – 11.2*3.22 –7.2k0.7 –7.9+1.0
LLT 50 (“C) –1.9 –7.9 >– 1.03 –2.0
Sample size for SCP determinationare 21, 18, 19 and 25 from top to bottomand those for LLT 50 are reported in the caption for Fig. 2. ‘ ND, non-diapause;D, diapause. 2 Significantly lower than the non-diapausing individuals (Aspin– Welch method, t=5.42,p< O.05). 3LLT 50 could not be calculated,because of the relatively small number of survivors.
DIAPAUSEAND COLD HARDINESSIN SPIDER
100
o—
0 —0
—0
—
273
o \
(A)
o
●
\ \
Diapausing o
Non-diapausing
●
\ \
o \
“\e
0 -1
-2
-3
-4
-5
-6
-7
-8
o -9
Temperature c)
100o
T > .
z
.-> >
( (B)
50 “
\
0
Diapausing
9 u) \
o-
0,
0 \
● \N;n-dia*a”sing
r -1
-2
-3
-4
0 , -5
Temperature c) FIGURE2. Survivorshipof (A) the cool-temperateand (B) the subtropicalpopulationof A. tepidariorum after 48h expsure to various temperature regimes between – 1 and –9”C. Sample size ranged from 7 to 28 individualsper treatment.
This means that the underlying mechanisms regulating the supercoolingability and chilling tolerance are different from each other, as suggestedfor other freeze-avoiding arthropod species (e.g. Lee and Denlinger, 1985; Bennett and Lee, 1989; Pullin et al., 1991; Tanaka and Udagawa, 1993).It has been demonstratedthat the SCP depression of this spider is achieved partly by cessation of feeding activity and excretion or inactivation of the gut ice nucleators (Tanaka, 1994, 1996),while the LLT 50 depression is correlated with the concentration of scyllo-inositoland myo-inositol,possible cryoprotectants (Tanaka, 1995). Secondly, the relationshipbetween diapause and cold hardinesschangeswith locality.As summarizedin Table
2, diapause spiders of the cool-temperate spiders increased both chilling tolerance and supercooling ability,while those of the subtropicalspidersenhanced the chillingtoleranceonly. This may reflecta local difference in winter climates. In Sapporo,located in the cool-temperateregion, winter temperaturesfrequently fall below the level of the summer SCP and LLT 50 (Tanaka, 1993), so that reduction in SCP and LLT 50 prior to the winter is essential for winter survival of this spider. In Naha, located in the subtropicalregion, winter temperaturesoccasionallygo down to around +7”C, but no frost occurs (Tanaka, 1996).Under such mild winter conditions,the overwinteringspiders are normally never exposed to a subzero temperature, thus they would not
KAZUHIROTANAKA
274
TABLE2. Relationshipof diapause, chillingtolerance and supercooling ability in A. tepidariorum at different climatic zones Climatic zones
Diapause
Chilling tolerance
Supercooling ability
Cool-temperate Subtropical Tropical
+ + ?
+ + ?
+ — ?
+, Presence; –, absence.
need to lower the SCP for overwintering. Unlike the SCP, LLT 50 levels are lowered in diapause individuals of the subtropicalspiders(Table 1). At present,however, the adaptivesignificanceof this phenomenonis not clear. The present results demonstrated that non-diapause nymphs suffered a higher rate of mortality after chilling at –1°C than did their diapausecounterparts(Fig. 2), but winter temperatures in Naha never drop to such a low level. Afier 48h exposure to +7”C, which is the lowest temperatureever recorded in Naha, no mortalityoccurred in non-diapause nymphs of the Naha population (K. Tanaka, unpublished observations). Although tirther confirmationis necessary, it is possiblethat the observed increase in chilling tolerance in the subtropical population is a byproduct associated with the induction of diapause. Such a physiologicaltrait, however, may provide a mechanism which enables the subtropicalpopulation to explore more northern climatic regions. A. tepidariorum is considered to be of tropical origin (Levi, 1967) and the subtropicaldiapause is likely to be an ancestral type of the temperate diapause (Tanaka, 1992).If this scenario is correct, it would follow that the ancestral diapause of this spider evolved with chilling tolerance, but without an ability to lower the SCP. The latter ability would have been evolved secondarilyas the spiders expanded their distribution to the temperate regions(Tanaka, 1996).Spidersexpandingfrom the tropical to the temperate regions would first encounter a problem associated with chilling or non-freezing low temperaturerather than freezingtemperature.In the early phase of their movement to the north, therefore, natural selection may act to reduce winter mortality by improving chilling tolerance. Under such circumstances, the ability to increase chilling tolerance in association with diapause induction would gain an adaptive value, and would be reinforcedthroughnatural selection.As spiders expand tiwthernorth, they would require a mechanismto survive fhrther low temperatures,which would create an opportunity for them to evolve an ability to lower the
SCP. This study is the first to characterizea differentialgeographic profile in the relationshipbetween chilling tolerance and supercooling ability in relation to diapause induction. It has demonstratedthat the evolutionof cold
hardiness in A. tepidariorum involves at least several steps in association with diapause. How commonly a similar phenomenon is seen among other invertebrate species that have expanded their distribution from the tropical to the temperate regions is not known. It is necessary to examine many more species to evaluate the generality of the above phenomenon. REFERENCES Bale J. S. (1993) Classes of insect cold hardiness. Furrct Ecof. 7, 751-753. Bennett L. E. and Lee R. E. (1989) Simulatedwinter to summer transition in diapausing adults of the lady beetle (Hippodamia convergent): supercoolingpoint is not indicativeof cold hardiness. Physiol. Entomol. 14, 361-367.
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Acknowledgements—I thank K. Shimada (Institute of Low Tempera-
ture Science, HokkaidoUniversity) and S. Tanaka (National Institute of Sericultural and Entomological Science) for reading the original manuscriptand H. Abe and Y. Saito (Facultyof Agriculture,Hokkaido University) for allowing me to use experimental facilities in the Laboratory of Applied Zoology. This research was in part supported by the JSPS Fellowshipfor Japanese Junior Scientists.