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Tissue distribution of the ice-nucleating agents in larvae of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)
CRYOBIOLOGY
28, 376-381 (1991)
Tissue Distribution of the Ice-Nucleating Agents in Larvae of the Rice Stem Borer, Chile suppressalis Walker (Lepidoptera: Pyralidae) HISAAKI TSUMUKI AND HARUYOSHI KONNO Research Institute for Bioresouces, Okayama University, Kurashiki 710, Japan To investigate the tissue distribution of ice-nucleating agents in the rice stem borer, Chilo suppressalis Walker, the crystallization temperatures of the whole bodies and individual tissues of nondiapausing and diapausing larvae were measured. In nondiapausing mature larvae the crystallization temperature of the gut with its contents was the highest, being about - 8”C, showing that a freezing site is present in the gut. As food particles in the alimentary canal of hibernating larvae were excreted in autumn, the larval supercooling capacity increased with lowering crystallization temperature of the gut. In diapausing larvae the crystallization temperatures of the muscle and epidermis were the highest, being above - 15”C, which is similar to that of the whole larvae, and the hemolymph crystallization temperature was the lowest, being below - 25°C. Furthermore, the crystallization temperatures of the nervous system, trachea, silk gland, salivary gland, and ovary were below - 2O”C, which was equivalent to those of 0.9% NaCl solution. Consequently, in diapausing larvae a primary site of freezing is present in the muscle and epidermis, indicating that the potent ice-nucleating agents exist in these tissues. However, since the epidermis could not be completely divided from the muscle, it was not concluded whether the potent ice-nucleating agents existed in the epidermis or not. 8 1991 Academic press, Inc
To survive extremely low temperatures in winter, insects utilize two main strategies, freezing tolerance and cold hardiness (freezing intolerance). Since the freezing intolerant species can not withstand the harmful effects of freezing, they avoid ice formation by the depression of crystallization temperature in winter (2, 14, 16). Most of them produce antifreeze substances, which depress the freezing point in water, and remove the ice-nucleating agents in their bodies (4). However, the freezing tolerant species can tolerate the formation of ice crystals in their bodies and still survive. Some of the species appear to freeze at high temperatures below zero, even when they have high concentrations of the antifreeze substances in their bodies (9). In general, extensive supercooling often induces lethal intracellular ice formation (1). Ice-nu-
Received June 23, 1990; accepted September 28, 1990.
cleating agents prevent extensive supercooling and induce ice formation at relatively high subzero temperatures so as to prevent intracellular ice formation (5, 7, 8, 22, 23).
In previous reports (19, 20), we demonstrated that larvae of the rice stem borer, Chilo suppressalis, accumulated glycerol in hemolymph in autumn and winter and, consequently, acquired a higher freezing tolerance. These results support the hypothesis that an increase of the larval freezing tolerance is correlated with glycerol production. Here, we report the evidence that crystallization of hibernating larvae occurs at relatively high subzero temperature. To elucidate the distribution of potent ice-nucleating agents, we have further attempted to examine the crystallization temperatures in the whole bodies and individual tissues of nondiapausing and diapausing larvae of the rice stem borer and thereby specify the tissue distributions of the ice-nucleating agents. 376
OOll-2240/91 $3.00 CopyQht All rights
Q 1991 by Academic Press, Inc. of reproduction in any form reserved.
TISSUE
DISTRIBUTION
MATERIALS
AND
OF ICE-NUCLEATING
METHODS
Mature larvae of the rice stem borer reared on rice seedlings at 25°C under long day length (16L, 8D) were used as nondiapausing insects (20). To study the effect of starvation on the crystallization temperatures, mature larvae were kept in a petri dish (9 cm diam. x 2 cm ht) at 25°C under a long day length without food and were used as needed. Hibernating larvae were collected monthly from the rice stems in fields. Since the larvae entered diapause at the end of November and broke at the beginning to middle of February in fields, the larvae collected in January were used as diapausing insect. To eliminate any surface ice-nucleating agents and to prevent the tissues from contamination with the agents during dissection, larvae were washed three or four times with distilled water. After the washed larvae were dried on filter paper, hemolymph issued by puncturing the larvae in the dorsum with a fine insect pin was collected directly into glass capillary tubes (60 x 1.1 mm id) and sandwiched between two layers of paraffin oil (24). After drawing hemolymph, the larvae were dissected in 0.9% NaCl solution and then divided into fat body, gut, and the remaining tissues (carcass). The carcass was further divided into trachea, silk gland, salivary gland, ovary, nervous system (nerve cords with some segmental ganglions), muscle, and epidermis. The dissected individual tissues were also sandwiched with the NaCl solution between two layers of the oil in capillary tubes. The crystallization temperatures were measured by a thermoelectric technique. Whole larvae and capillary tubes containing the individual tissues were attached to 30-gauge copper-constantan thermocouples connected to a multichannel temperature recorder (Takeda Riken, Japan) and then cooled at a rate of about l”C/min until frozen. The crystallization temperature was determined as the temperature at which a rapid increase in tempera-
AGENTS
IN
THE
RICE
STEM
BORER
377
ture occurred due to the release of the latent heat of fusion. Crystallization temperatures of 12 samples could be measured simultaneously by the recorder. To collect the frass, nondiapausing mature larvae were transferred into a petri dish (9 cm diam X 2 cm ht) and then some frasses excreted by them were directly transferred into the capillary tubes containing 0.9% NaCl solution. The crystallization temperature of the frass was also measured. RESULTS
The crystallization temperatures of the whole bodies and individual tissues of nondiapausing mature larvae were measured (Table 1). The crystallization temperature of the hemolymph was lower than that of the other tissues tested, while the tissue having the highest crystallization temperature was the gut. Furthermore, the crystallization temperatures of the gut content and frass were high as well. There was no significant difference in the crystallization temperature between whole larvae ( - 7.7” TABLE 1 Crystallization Temperatures in the Tissues of Nondiapausing Mature Larvae of the Rice Stem Borer Tissue Hemolymph Fat body Gut Silk gland Salivary gland Trachea Nervous system ovary
Muscle Epidermis Whole body Gut content Frass
0.9% NaCl solution
Crystallization temperature (“C) -24.5 2 0.8 (lo)** - 18.9 f 0.8 (lo)** -8.4 ” 0.3 (10) -20.1 2 0.3 (7)** -23.6 -20.0 -22.4 -20.9
f k 2 +
0.7 0.5 0.7 0.3
(5)** @I)** (5)** (5)**
- 16.1 ? 0.3 (lo)** - 12.7 2 0.7 (lo)** -7.7 + 0.4 (10) -8.3 -6.5 -20.5
+ 0.4 (7)
2 0.1 (5) 2 0.6 (4)**
Note. All values are means + SE (N).
** Significantly different (Student I test) from the value of whole body, P < 0.01.
378
TSUMUKI
+- 0.4”C) and gut (- 8.4” + 0.3”C) or its content (-8.3” + 0.4”C). The crystallization temperatures of the nervous system, trachea, silk gland, salivary gland, and ovary were less than or equal to -2O”C, which was equivalent to those of the 0.9% NaCl solution, whereas the muscle and epidermis had relatively high crystallization temperatures. The effect of starvation on the supercooling capacities of nondiapausing mature larvae was studied (Fig. 1). The crystallization temperature remained constant for the first 2 days after starvation and, thereafter, lowered gradually. The crystallization temperatures of the whole larvae, hemolymph, fat body, and gut were measured during hibernation (Fig. 2). Except for the larvae collected from fields in October, the crystallization temperatures of hibernating larvae were at approximately constant levels. In diapausing larvae as well as in nondiapausing mature insects the crystallization temperature of the hemolymph appeared to be the lowest of all tissues tested. The crystallization temperature of the whole larvae was significantly higher than those of the hemolymph, fat body, and gut (haemolymph and fat body, P < 0.01; gut, P < 0.05). In the larvae collected in October, however, there was no significant difference in the crystallization temperature be-
G e
cl I
FIG. 1. Effect of starvation on the crystallization temperatures of nondiapausing mature larvae of the rice stem borer. Bars represent SE of mean (N). Significant differences (Student r test) were observed between Day 0 and Day 5 (P < 0.05), or Day IO (P c: 0.01).
AND
KONNO
0
i
0f -301
, OCt
NW
DBC
Jan
Feb
Mar
APr
May
Month
FIG. 2. Changes of the crystallization temperatures of whole larvae and tissues during hibernation of the rice stem borer. Bars represent SE of mean (IV). Except for the larvae collected in October, significant differences (Student t test, P < 0.05) were observed between gut and whole larvae during hibernation.
tween the gut (-9.7” 5 0.7”C) and whole body (- 8.5” + O.SOC).These results suggest that in diapausing larvae the freezing site would be distributed in the carcass. Therefore, the carcass of diapausing larvae was further divided and the crystallization temperatures of the individual tissues were measured (Table 2). The data show that the crystallization temperatures of the nervous system, silk gland, salivary gland, ovary, and trachea were below -2O”C, while the muscle and epidermis had crystallization temperatures of - 13.7” ? 0.8”C and - 14.8” * O.YC, respectively, which were compatible with the temperature of the whole larvae (I 13.8” -+ l.O’C). When the hemolymph was diluted with 0.9% NaCl solution, the crystallization temperature was raised to the equivalent of the NaCl solution (Table 3). DISCUSSION
In the present study it is demonstrated for the first time that the muscle is identified as a primary site of freezing in diapausing larvae of the rice stem borer.
TISSUE
DISTRIBUTION
OF ICE-NUCLEATING
AGENTS
IN THE RICE STEM BORER
379
The variations in crystallization temperatures of the whole larvae are explained by those of the specific tissues. In diapausing larvae, lower crystallization temperatures Crystallization temperature (“C) Tissue of the hemolymph, fat body, and gut than those of the whole larvae show that the priHemolymph -26.3 f 0.3 (8)** mary site of freezing did not exist in these Fat body -20.9 + 1.1 (8)** Gut - 17.1 + 1.0 (8)* tissues. The muscle and epidermis had Silk gland -20.3 2 0.5 (7)** crystallization temperatures of - 13.7” + Salivary gland -22.7 * 0.5 (7)** 0.8”C and - 14.8” -+ 0.9”C, respectively, Trachea -20.1 ? 0.5 (8)** which were approximately equal to the Nervous system -22.5 2 0.2 (6)** temperature of the whole larvae (- 13.8” 2 ovary -22.0 2 0.6 (8)** Muscle - 13.7 ? 0.8 (8) l.O”C), indicating that the primary site of Epidermis - 14.8 ? 0.9 (8) freezing is possibly present in these tissues. Whole body - 13.8 ? 1.0 (24) In this experiment, however, isolated musGut content cle from the epidermis was obtained but the 0.9% NaCl solution -20.5 2 0.6 (4)** epidermis could not be separated comAll values are means 2 SE (IV). pletely from the muscle. Consequently it * Significantly different (Student t test) from the cannot be determined whether the crystalvalue of whole body. P < 0.05; **P < 0.01. lization temperature of the epidermis is due to the activity of the remaining muscle on The rice stem borer survives the winter the epidermis or ice-nucleating agents in in diapause as mature larvae in the rice the epidermis. In nondiapausing larvae, stems. Although an increase in the freezing however, a significantly higher crystallizatolerance (maximal low temperature toler- tion temperature of the epidermis (- 12.7” ance limit of below -30°C) of hibernating ? 0.7”C) than that of the muscle (16.1” -+ larvae seemed to be due to high glycerol 0.3”C) (Student t test, P < 0.05) shows that content in hemolymph (19), the larvae had the temperature of the epidermis is affected mean crystallization temperatures above by the activity of remaining ice-nucleators - 15°C during hibernation. It is therefore attached on a surface of the tissue (3, 17). indicated that the larvae are classified as a The ice-nucleating active agents confreezing tolerant species. Nondiapausing tained in insects can be classified into two mature larvae could survive freezing, but types, exogenous and endogenous agents. their freezing tolerance limit, being about The exogenous agents are distributed in gut - IO”C, was lower than diapausing individcontents (6, 12, 13, 15) and on body surface uals (19). (3, 17), while a notable site of the endogenous type appears to be in hemolymph (22). TABLE 3 In rice stem borer larvae the exogenous and The Crystallization Temperatures in the Hemolymph endogenous ice-nucleating active agents Diluted Repeatedly by 0.9% NaCl Solution are present in the alimentary canal or its Crystallization contents and the muscle (and epidermis), temperature CC) Dilution respectively. When food particles present loo 10 factor 0 in the alimentary canal were excreted at the -20.9 f 0.8’ -21.2 f 0.2* -25.4 + 0.6 end of October to the beginning of November, the supercooling capacity of the whole Note. Each value is the mean (mean + SE) of four replicate samples. larvae decreased markedly. Consequently, * Significantly different (Student f test) from the value for in diapausing larvae, the ice-nucleating acdilution factor 0, P < 0.05. TABLE 2 Crystallization Temperatures in the Tissues of Diapausing Larvae of the Rice Stem Borer
380
TSUMUKI
tive agents present in the muscle (and epidermis) and more potent than those in the gut and fat body and, therefore, they may play an important role during winter. The crystallization temperature of muscle in diapausing larvae was higher than that in nondiapausing mature larvae, showing that some new potential ice-nucleating agents may be synthesized with the initiation of diapause, but the nucleators are present in the tissue of nondiapausing larvae. Further studies are currently under way to determine the distribution and to characterize the nucleating agents in diapausing and nondiapausing larvae. Although the ice-nucleating active agents were present in the muscle of nondiapausing larvae as well as diapausing larvae, the activities would be masked by the gut agents, which have a much higher crystallization temperature than the muscle agents. The mean crystallization temperatures of the silk gland, salivary gland, nervous system, trachea, ovary, and hemolymph immersed in 0.9% NaCl solution were in the range of - 20” to - 24°C which were equivalent to those of the control solution. Although an accurate crystallization temperature of these tissues could not be measured in these experiments, potential ice-nucleating agents would presumably absent. Although some freezing tolerant insects possess ice-nucleating agents in their hemolymph (8, 22, 23), the ice-nucleating agents are absent from the hemolymph of rice stem borer larvae as well as other freeze tolerant species (4, 10, 11). An accumulation of cryoprotective substances depresses crystallization temperatures (21). In the rice stem borer, although there was a shift in the levels of glycerol and trehalose in the hemolymph as cryoprotective substances during hibernation (19, 20), the mean hemolymph crystallization temperatures were approximately constant during hibernation, revealing that the
AND
KONNO
temperatures may be influenced by factor(s) other than the solute concentrations, possibly a lack of ice-nucleating activity. Although it has been shown in the lady beetle that ingestion of ice-nucleating active bacteria elevates the crystallization temperature (18), the bacteria may play a less important role in diapausing larvae of the rice stem borer which excretes its gut contents before diapause. SUMMARY
The crystallization temperatures of the whole bodies and individual tissues of nondiapausing and diapausing larvae were measured. In non diapausing mature larvae the crystallization temperature of the gut with its contents was higher than that of the other tissues tested, showing that freezing site is present in the gut. As food particles in the alimentary canal of hibernating larvae were excreted in autumn, the larval supercooling capacity decreased with lowering crystallization temperature of the gut. In diapausing larvae the crystallization of the muscle and epidermis occurred at the highest temperature, being above - WC, which is similar to that of the whole larvae and the hemolymph crystallization temperature was the lowest, being below - 25°C. Furthermore, the crystallization temperatures of the nervous system, trachea, silk gland, salivary gland, and ovary were below -20°C which was equivalent to those of the 0.9% NaCl solution. Consequently, in diapausing larvae a primary site of freezing is present in the muscle and epidermis, indicating that the potent ice-nucleating agents exist in these tissues. However, since the epidermis could not be completely divided from the muscle, it is not yet known whether the potent ice-nucleating agents existed in the epidermis of diapausing larvae or not. ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid
TISSUE DISTRIBUTION
OF ICE-NUCLEATING
for Scientific Research (No. 01560053)and a Grant for Special Project Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. Asahina, E. Frost resistance in insects. Adv. Znsect Physiol. 6, 149 (1%9). 2. Bale, J. S. Insect cold hardiness: Freezing and supercooling-an ecophysiological perspective. J. Insect Physid. 33, 899-908 (1987). 3. Bale, J. S., Hansen, T. N., and Baust, J. G. Nucleators and sites of nucleation in the freeze tolerant larvae of the gallfly Eurosta solidaginis (Fitch). J. Insect Physiol. 35, 291-298 (1989). 4. Baust, J. G., and Rojas, R. R. Review-insect cold hardiness: Facts and fancy. J. Insect Physiol. 31, 755-759 (1985). 5. Baust, J. G., and Zachariassen, K. E. Seasonally active cell matrix associated ice nucleators in an insect. Cryo Lett. 4, 65-71 (1983). 6. Block, W., and Zettel, J. Cold hardiness of some Alpine Collemhola. Ecol. En?. 5, l-9 (1980). 7. Duman, J. G., and Patterson, J. L. The role of ice nucleators in the frost tolerance of overwintering queens of the bold faced hamet. Camp. Biothem. Physiol. 59A, 69-72 (1978). 8. Duman, J. G., and Horwath, K. L. The role of hemolymph proteins in the cold tolerance of insects. Annu. Rev. Physiol. 45, 261-270 (1983). 9. Lee, R. E., Jr., Zachariassen, K. E., and Baust, J. G. Effect of cryoprotectants on the activity of hemolymph nucleating agents in physical solutions. Cryobiology 18, 511-514 (1981). 10. Miller, K. Cold-hardiness strategies of some adult and inmature insects overwintering in interior Alaska. Comp. Biochem. Physiol. 73A, 595-604 (1982). 11. Ring, R. A. Freezing-tolerant insects with low supercooling points. Comp. Biochem. Physiol. A 73, 605412 (1982). 12. Salt, R. W. Factors influencing nucleation in supercooled insects. Can. J. Zoo/. 44, 117-133 (1966). 13. Salt, R. W. Location and quantitative aspects of ice nucleation in insects. Can. J. Zool. 46, 329333 (1968).
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14. S@mme, L. Supercooling and winter survival in terrestrial arthropods. Camp. Biochem. Physiol. A 73, 519-543 (1982). 15. S@mme, L., and Conradi-Larsen, E. M. Coldhardiness of collembolans and oribatid mites from windswept mountain ridges. Oikos 29, 127-132 (1977). 16. Storey, K. B., and Storey, J. M. Freeze tolerance in animals. Physiol. Rev. 68, 27-84 (1988). 17. Strong-Gunderson, J. M., Lee, R. E., Jr., and Lee, M. R. Ice-nucleating bacteria promote transcuticular nucleation in insects. Cryobiology 25, 551 (1989). 18. Strong-Gunderson, J. M., Lee, R. E., Jr., Lee, M. R., and Riga, T. J. Ingestion of icenucleating active bacteria increases the supercooling point of the lady beetle Hippodumia convergens. J. Insect Physiol. 36, 153-157 Wm. 19. Tsumuki, H. Environmental adaptations of the rice stem borer, Chile suppressalis and the blue alfalfa aphid, Acyrthosiphon kondoi to seasonal fluctuations. In “Advance in Invertebrate Reproduction” (Hoshi, M., and Yamashita, O., Eds.), Vol. 5, pp. 273-278. Elsevier Science Publishers, Amsterdam, 1990. 20. Tsumuki, H., and Kanehisa, K. Carbohydrate content and oxygen uptake in larvae of the rice stem borer, Chile suppressalis Walker. Ber. Ohara Znst. Landwirt. Biol. Okayama Univ. 17, 95-110 (1978). 21. Zachariassen, K. E. The role of polyols and nucleating agents in cold-hardy beetles. J. Camp. Physiol. 140, 227-234 (1980). 22. Zachariassen, K. E. Nucleating agents in coldhardy insects. Comp. Biochem. Physiol. A 73, 557-562 (1982). 23. Zachariassen, K. E., and Hammel, H. T. Nucleating agents in the haemolymph of insects tolerant to freezing. Nature (London) 262,285-287 (1976). 24. Zachariassen, K. E., Baust, J. G., and Lee, R. E., Jr. A method for quantitative determination of ice nucleating agents in insect hemolymph. Cryobiology 19, 180-184 (1982).
28, 376-381 (1991)
Tissue Distribution of the Ice-Nucleating Agents in Larvae of the Rice Stem Borer, Chile suppressalis Walker (Lepidoptera: Pyralidae) HISAAKI TSUMUKI AND HARUYOSHI KONNO Research Institute for Bioresouces, Okayama University, Kurashiki 710, Japan To investigate the tissue distribution of ice-nucleating agents in the rice stem borer, Chilo suppressalis Walker, the crystallization temperatures of the whole bodies and individual tissues of nondiapausing and diapausing larvae were measured. In nondiapausing mature larvae the crystallization temperature of the gut with its contents was the highest, being about - 8”C, showing that a freezing site is present in the gut. As food particles in the alimentary canal of hibernating larvae were excreted in autumn, the larval supercooling capacity increased with lowering crystallization temperature of the gut. In diapausing larvae the crystallization temperatures of the muscle and epidermis were the highest, being above - 15”C, which is similar to that of the whole larvae, and the hemolymph crystallization temperature was the lowest, being below - 25°C. Furthermore, the crystallization temperatures of the nervous system, trachea, silk gland, salivary gland, and ovary were below - 2O”C, which was equivalent to those of 0.9% NaCl solution. Consequently, in diapausing larvae a primary site of freezing is present in the muscle and epidermis, indicating that the potent ice-nucleating agents exist in these tissues. However, since the epidermis could not be completely divided from the muscle, it was not concluded whether the potent ice-nucleating agents existed in the epidermis or not. 8 1991 Academic press, Inc
To survive extremely low temperatures in winter, insects utilize two main strategies, freezing tolerance and cold hardiness (freezing intolerance). Since the freezing intolerant species can not withstand the harmful effects of freezing, they avoid ice formation by the depression of crystallization temperature in winter (2, 14, 16). Most of them produce antifreeze substances, which depress the freezing point in water, and remove the ice-nucleating agents in their bodies (4). However, the freezing tolerant species can tolerate the formation of ice crystals in their bodies and still survive. Some of the species appear to freeze at high temperatures below zero, even when they have high concentrations of the antifreeze substances in their bodies (9). In general, extensive supercooling often induces lethal intracellular ice formation (1). Ice-nu-
Received June 23, 1990; accepted September 28, 1990.
cleating agents prevent extensive supercooling and induce ice formation at relatively high subzero temperatures so as to prevent intracellular ice formation (5, 7, 8, 22, 23).
In previous reports (19, 20), we demonstrated that larvae of the rice stem borer, Chilo suppressalis, accumulated glycerol in hemolymph in autumn and winter and, consequently, acquired a higher freezing tolerance. These results support the hypothesis that an increase of the larval freezing tolerance is correlated with glycerol production. Here, we report the evidence that crystallization of hibernating larvae occurs at relatively high subzero temperature. To elucidate the distribution of potent ice-nucleating agents, we have further attempted to examine the crystallization temperatures in the whole bodies and individual tissues of nondiapausing and diapausing larvae of the rice stem borer and thereby specify the tissue distributions of the ice-nucleating agents. 376
OOll-2240/91 $3.00 CopyQht All rights
Q 1991 by Academic Press, Inc. of reproduction in any form reserved.
TISSUE
DISTRIBUTION
MATERIALS
AND
OF ICE-NUCLEATING
METHODS
Mature larvae of the rice stem borer reared on rice seedlings at 25°C under long day length (16L, 8D) were used as nondiapausing insects (20). To study the effect of starvation on the crystallization temperatures, mature larvae were kept in a petri dish (9 cm diam. x 2 cm ht) at 25°C under a long day length without food and were used as needed. Hibernating larvae were collected monthly from the rice stems in fields. Since the larvae entered diapause at the end of November and broke at the beginning to middle of February in fields, the larvae collected in January were used as diapausing insect. To eliminate any surface ice-nucleating agents and to prevent the tissues from contamination with the agents during dissection, larvae were washed three or four times with distilled water. After the washed larvae were dried on filter paper, hemolymph issued by puncturing the larvae in the dorsum with a fine insect pin was collected directly into glass capillary tubes (60 x 1.1 mm id) and sandwiched between two layers of paraffin oil (24). After drawing hemolymph, the larvae were dissected in 0.9% NaCl solution and then divided into fat body, gut, and the remaining tissues (carcass). The carcass was further divided into trachea, silk gland, salivary gland, ovary, nervous system (nerve cords with some segmental ganglions), muscle, and epidermis. The dissected individual tissues were also sandwiched with the NaCl solution between two layers of the oil in capillary tubes. The crystallization temperatures were measured by a thermoelectric technique. Whole larvae and capillary tubes containing the individual tissues were attached to 30-gauge copper-constantan thermocouples connected to a multichannel temperature recorder (Takeda Riken, Japan) and then cooled at a rate of about l”C/min until frozen. The crystallization temperature was determined as the temperature at which a rapid increase in tempera-
AGENTS
IN
THE
RICE
STEM
BORER
377
ture occurred due to the release of the latent heat of fusion. Crystallization temperatures of 12 samples could be measured simultaneously by the recorder. To collect the frass, nondiapausing mature larvae were transferred into a petri dish (9 cm diam X 2 cm ht) and then some frasses excreted by them were directly transferred into the capillary tubes containing 0.9% NaCl solution. The crystallization temperature of the frass was also measured. RESULTS
The crystallization temperatures of the whole bodies and individual tissues of nondiapausing mature larvae were measured (Table 1). The crystallization temperature of the hemolymph was lower than that of the other tissues tested, while the tissue having the highest crystallization temperature was the gut. Furthermore, the crystallization temperatures of the gut content and frass were high as well. There was no significant difference in the crystallization temperature between whole larvae ( - 7.7” TABLE 1 Crystallization Temperatures in the Tissues of Nondiapausing Mature Larvae of the Rice Stem Borer Tissue Hemolymph Fat body Gut Silk gland Salivary gland Trachea Nervous system ovary
Muscle Epidermis Whole body Gut content Frass
0.9% NaCl solution
Crystallization temperature (“C) -24.5 2 0.8 (lo)** - 18.9 f 0.8 (lo)** -8.4 ” 0.3 (10) -20.1 2 0.3 (7)** -23.6 -20.0 -22.4 -20.9
f k 2 +
0.7 0.5 0.7 0.3
(5)** @I)** (5)** (5)**
- 16.1 ? 0.3 (lo)** - 12.7 2 0.7 (lo)** -7.7 + 0.4 (10) -8.3 -6.5 -20.5
+ 0.4 (7)
2 0.1 (5) 2 0.6 (4)**
Note. All values are means + SE (N).
** Significantly different (Student I test) from the value of whole body, P < 0.01.
378
TSUMUKI
+- 0.4”C) and gut (- 8.4” + 0.3”C) or its content (-8.3” + 0.4”C). The crystallization temperatures of the nervous system, trachea, silk gland, salivary gland, and ovary were less than or equal to -2O”C, which was equivalent to those of the 0.9% NaCl solution, whereas the muscle and epidermis had relatively high crystallization temperatures. The effect of starvation on the supercooling capacities of nondiapausing mature larvae was studied (Fig. 1). The crystallization temperature remained constant for the first 2 days after starvation and, thereafter, lowered gradually. The crystallization temperatures of the whole larvae, hemolymph, fat body, and gut were measured during hibernation (Fig. 2). Except for the larvae collected from fields in October, the crystallization temperatures of hibernating larvae were at approximately constant levels. In diapausing larvae as well as in nondiapausing mature insects the crystallization temperature of the hemolymph appeared to be the lowest of all tissues tested. The crystallization temperature of the whole larvae was significantly higher than those of the hemolymph, fat body, and gut (haemolymph and fat body, P < 0.01; gut, P < 0.05). In the larvae collected in October, however, there was no significant difference in the crystallization temperature be-
G e
cl I
FIG. 1. Effect of starvation on the crystallization temperatures of nondiapausing mature larvae of the rice stem borer. Bars represent SE of mean (N). Significant differences (Student r test) were observed between Day 0 and Day 5 (P < 0.05), or Day IO (P c: 0.01).
AND
KONNO
0
i
0f -301
, OCt
NW
DBC
Jan
Feb
Mar
APr
May
Month
FIG. 2. Changes of the crystallization temperatures of whole larvae and tissues during hibernation of the rice stem borer. Bars represent SE of mean (IV). Except for the larvae collected in October, significant differences (Student t test, P < 0.05) were observed between gut and whole larvae during hibernation.
tween the gut (-9.7” 5 0.7”C) and whole body (- 8.5” + O.SOC).These results suggest that in diapausing larvae the freezing site would be distributed in the carcass. Therefore, the carcass of diapausing larvae was further divided and the crystallization temperatures of the individual tissues were measured (Table 2). The data show that the crystallization temperatures of the nervous system, silk gland, salivary gland, ovary, and trachea were below -2O”C, while the muscle and epidermis had crystallization temperatures of - 13.7” ? 0.8”C and - 14.8” * O.YC, respectively, which were compatible with the temperature of the whole larvae (I 13.8” -+ l.O’C). When the hemolymph was diluted with 0.9% NaCl solution, the crystallization temperature was raised to the equivalent of the NaCl solution (Table 3). DISCUSSION
In the present study it is demonstrated for the first time that the muscle is identified as a primary site of freezing in diapausing larvae of the rice stem borer.
TISSUE
DISTRIBUTION
OF ICE-NUCLEATING
AGENTS
IN THE RICE STEM BORER
379
The variations in crystallization temperatures of the whole larvae are explained by those of the specific tissues. In diapausing larvae, lower crystallization temperatures Crystallization temperature (“C) Tissue of the hemolymph, fat body, and gut than those of the whole larvae show that the priHemolymph -26.3 f 0.3 (8)** mary site of freezing did not exist in these Fat body -20.9 + 1.1 (8)** Gut - 17.1 + 1.0 (8)* tissues. The muscle and epidermis had Silk gland -20.3 2 0.5 (7)** crystallization temperatures of - 13.7” + Salivary gland -22.7 * 0.5 (7)** 0.8”C and - 14.8” -+ 0.9”C, respectively, Trachea -20.1 ? 0.5 (8)** which were approximately equal to the Nervous system -22.5 2 0.2 (6)** temperature of the whole larvae (- 13.8” 2 ovary -22.0 2 0.6 (8)** Muscle - 13.7 ? 0.8 (8) l.O”C), indicating that the primary site of Epidermis - 14.8 ? 0.9 (8) freezing is possibly present in these tissues. Whole body - 13.8 ? 1.0 (24) In this experiment, however, isolated musGut content cle from the epidermis was obtained but the 0.9% NaCl solution -20.5 2 0.6 (4)** epidermis could not be separated comAll values are means 2 SE (IV). pletely from the muscle. Consequently it * Significantly different (Student t test) from the cannot be determined whether the crystalvalue of whole body. P < 0.05; **P < 0.01. lization temperature of the epidermis is due to the activity of the remaining muscle on The rice stem borer survives the winter the epidermis or ice-nucleating agents in in diapause as mature larvae in the rice the epidermis. In nondiapausing larvae, stems. Although an increase in the freezing however, a significantly higher crystallizatolerance (maximal low temperature toler- tion temperature of the epidermis (- 12.7” ance limit of below -30°C) of hibernating ? 0.7”C) than that of the muscle (16.1” -+ larvae seemed to be due to high glycerol 0.3”C) (Student t test, P < 0.05) shows that content in hemolymph (19), the larvae had the temperature of the epidermis is affected mean crystallization temperatures above by the activity of remaining ice-nucleators - 15°C during hibernation. It is therefore attached on a surface of the tissue (3, 17). indicated that the larvae are classified as a The ice-nucleating active agents confreezing tolerant species. Nondiapausing tained in insects can be classified into two mature larvae could survive freezing, but types, exogenous and endogenous agents. their freezing tolerance limit, being about The exogenous agents are distributed in gut - IO”C, was lower than diapausing individcontents (6, 12, 13, 15) and on body surface uals (19). (3, 17), while a notable site of the endogenous type appears to be in hemolymph (22). TABLE 3 In rice stem borer larvae the exogenous and The Crystallization Temperatures in the Hemolymph endogenous ice-nucleating active agents Diluted Repeatedly by 0.9% NaCl Solution are present in the alimentary canal or its Crystallization contents and the muscle (and epidermis), temperature CC) Dilution respectively. When food particles present loo 10 factor 0 in the alimentary canal were excreted at the -20.9 f 0.8’ -21.2 f 0.2* -25.4 + 0.6 end of October to the beginning of November, the supercooling capacity of the whole Note. Each value is the mean (mean + SE) of four replicate samples. larvae decreased markedly. Consequently, * Significantly different (Student f test) from the value for in diapausing larvae, the ice-nucleating acdilution factor 0, P < 0.05. TABLE 2 Crystallization Temperatures in the Tissues of Diapausing Larvae of the Rice Stem Borer
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TSUMUKI
tive agents present in the muscle (and epidermis) and more potent than those in the gut and fat body and, therefore, they may play an important role during winter. The crystallization temperature of muscle in diapausing larvae was higher than that in nondiapausing mature larvae, showing that some new potential ice-nucleating agents may be synthesized with the initiation of diapause, but the nucleators are present in the tissue of nondiapausing larvae. Further studies are currently under way to determine the distribution and to characterize the nucleating agents in diapausing and nondiapausing larvae. Although the ice-nucleating active agents were present in the muscle of nondiapausing larvae as well as diapausing larvae, the activities would be masked by the gut agents, which have a much higher crystallization temperature than the muscle agents. The mean crystallization temperatures of the silk gland, salivary gland, nervous system, trachea, ovary, and hemolymph immersed in 0.9% NaCl solution were in the range of - 20” to - 24°C which were equivalent to those of the control solution. Although an accurate crystallization temperature of these tissues could not be measured in these experiments, potential ice-nucleating agents would presumably absent. Although some freezing tolerant insects possess ice-nucleating agents in their hemolymph (8, 22, 23), the ice-nucleating agents are absent from the hemolymph of rice stem borer larvae as well as other freeze tolerant species (4, 10, 11). An accumulation of cryoprotective substances depresses crystallization temperatures (21). In the rice stem borer, although there was a shift in the levels of glycerol and trehalose in the hemolymph as cryoprotective substances during hibernation (19, 20), the mean hemolymph crystallization temperatures were approximately constant during hibernation, revealing that the
AND
KONNO
temperatures may be influenced by factor(s) other than the solute concentrations, possibly a lack of ice-nucleating activity. Although it has been shown in the lady beetle that ingestion of ice-nucleating active bacteria elevates the crystallization temperature (18), the bacteria may play a less important role in diapausing larvae of the rice stem borer which excretes its gut contents before diapause. SUMMARY
The crystallization temperatures of the whole bodies and individual tissues of nondiapausing and diapausing larvae were measured. In non diapausing mature larvae the crystallization temperature of the gut with its contents was higher than that of the other tissues tested, showing that freezing site is present in the gut. As food particles in the alimentary canal of hibernating larvae were excreted in autumn, the larval supercooling capacity decreased with lowering crystallization temperature of the gut. In diapausing larvae the crystallization of the muscle and epidermis occurred at the highest temperature, being above - WC, which is similar to that of the whole larvae and the hemolymph crystallization temperature was the lowest, being below - 25°C. Furthermore, the crystallization temperatures of the nervous system, trachea, silk gland, salivary gland, and ovary were below -20°C which was equivalent to those of the 0.9% NaCl solution. Consequently, in diapausing larvae a primary site of freezing is present in the muscle and epidermis, indicating that the potent ice-nucleating agents exist in these tissues. However, since the epidermis could not be completely divided from the muscle, it is not yet known whether the potent ice-nucleating agents existed in the epidermis of diapausing larvae or not. ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid
TISSUE DISTRIBUTION
OF ICE-NUCLEATING
for Scientific Research (No. 01560053)and a Grant for Special Project Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. Asahina, E. Frost resistance in insects. Adv. Znsect Physiol. 6, 149 (1%9). 2. Bale, J. S. Insect cold hardiness: Freezing and supercooling-an ecophysiological perspective. J. Insect Physid. 33, 899-908 (1987). 3. Bale, J. S., Hansen, T. N., and Baust, J. G. Nucleators and sites of nucleation in the freeze tolerant larvae of the gallfly Eurosta solidaginis (Fitch). J. Insect Physiol. 35, 291-298 (1989). 4. Baust, J. G., and Rojas, R. R. Review-insect cold hardiness: Facts and fancy. J. Insect Physiol. 31, 755-759 (1985). 5. Baust, J. G., and Zachariassen, K. E. Seasonally active cell matrix associated ice nucleators in an insect. Cryo Lett. 4, 65-71 (1983). 6. Block, W., and Zettel, J. Cold hardiness of some Alpine Collemhola. Ecol. En?. 5, l-9 (1980). 7. Duman, J. G., and Patterson, J. L. The role of ice nucleators in the frost tolerance of overwintering queens of the bold faced hamet. Camp. Biothem. Physiol. 59A, 69-72 (1978). 8. Duman, J. G., and Horwath, K. L. The role of hemolymph proteins in the cold tolerance of insects. Annu. Rev. Physiol. 45, 261-270 (1983). 9. Lee, R. E., Jr., Zachariassen, K. E., and Baust, J. G. Effect of cryoprotectants on the activity of hemolymph nucleating agents in physical solutions. Cryobiology 18, 511-514 (1981). 10. Miller, K. Cold-hardiness strategies of some adult and inmature insects overwintering in interior Alaska. Comp. Biochem. Physiol. 73A, 595-604 (1982). 11. Ring, R. A. Freezing-tolerant insects with low supercooling points. Comp. Biochem. Physiol. A 73, 605412 (1982). 12. Salt, R. W. Factors influencing nucleation in supercooled insects. Can. J. Zoo/. 44, 117-133 (1966). 13. Salt, R. W. Location and quantitative aspects of ice nucleation in insects. Can. J. Zool. 46, 329333 (1968).
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