Juvenile hormone modulation of insect cold hardening: Ice-nucleating activity

CRYOBIOLOGY

24, 465-472 (1987)

Juvenile

Hormone

R. R. ROJAS,’

Modulation Ice-Nucleating

of Insect Cold Hardening: Activity

M. D. HAMILTON,

Institute of Low Temperature Biology, University *Center for Cryobiological Research, Department State University of New

AND J. G. BAUST*

of Houston-University of Biological Sciences, York, Binghamton, New

Park, Houston, Texas 77004, and University Center at Binghamton, York 13901

Data are presented offering the first evidence for probable endocrine involvement in the control of cold hardening in Eurosta solidaginis. Juvenile hormone (JH) deprivation experiments in which the corpora allata were removed by head ligation resulted in a loss of supercooling (SC) capacity in larvae collected over 2 years. This loss of supercooling capacity is indicative of synthesis of organismal pools of ice-nucleating agents (INA). Larval sensitivity to JH removal (ligation) on SC is seasonally dependent. For example, in 1983, larvae were most sensitive in October, secondarily so in September, and relatively insensitive in December regardless of acclimation temperature. While in 1984, larvae acclimated to + 5°C were most sensitive in November, secondarily so in October, and relatively insensitive in December, and larvae acclimated to + 15°C were most sensitive in October and December and least so in November. Supercooling point elevations as great as 7°C over controls were observed with maximal responses occurring within 1 day following ligation. In 1983, juvenile hormone replacement following ligation generally resulted in an expansion of supercooling capacity when compared to controls. As with ligation, the sensitivity to JH replacement on SC was seasonally dependent: September and October larvae being the most sensitive with December larvae being insensitive. Larvae collected in 1984 were given a greater dose of JH than those in the previous year and showed no significant change in SC over ligated-acetone controls. Hormone analog potentiation experiments in which unligated larvae collected in 1983 and acclimated to + 5°C were given methoprene resulted in depression of supercooling points for September and October larvae. JH titres appear to play an important role in the regulation of SC capacity in E. solidaginis larvae. 0 1987 Academic Press, Inc. INTRODUCTION

The third-instar larva of Eurosta sofidaginis overwinters and becomes freezing tolerant. Baust and Lee (3) reported that a southern population of E. solidaginis depresses the supercooling point from - 10.2 to - 14°C during autumn and early winter. This change is associated with a loss of icenucleating agents (INAs) and provides the third-instar larvae with a freezing avoidance strategy. That is, its “freezing point” is depressed well below any ambient low temperatures. Many freeze tolerant insects contain icenucleating agents which act antagonistically to antifreeze agents to induce freezing at relatively high subzero temperatures (13, 18). Those insects that are freezing intolerant generally lack INAs and supercool from - 12 to - 60°C. INAs are thought to confer freezing tolerance by inducing

Cold hardiness in insect species is associated with specific life stages in which the organism becomes responsive to environmental cues, i.e., temperature, photoperiod, and desiccation. Since insect development is under endocrine control, it is not improbable that induction of cold hardiness is also influenced by the endocrine system. Horwath and Duman (6) have shown that in the larvae of Dendvoides canadensis the production of antifreeze proteins is induced by juvenile hormone. Juvenile hormone (JH) is produced and stored in the corpora allata and has been found to induce and maintain diapause.

Received February 9, 1987; accepted June 8, 1987. I To whom reprint requests should be addressed. 465

001 I-2240187 $3.00 Copyright 0 1987 by Academic Presc, Inc. All rights of reproductmn in any form revzrvcd.

466

ROJAS.

HAMILTON,

freezing at relatively high subzero temperatures, precluding the formation of intracellular ice that would occur with freezing at lower temperatures. They vary in their occurrence and location between species and within different life stages within species (13). For example, INAs have been associated with gut contents (9-12, 14), hemolymph (13, 18), and the cell matrix (4). Hemolymph-borne INAs of freeze-tolerant insects are proteinaceous in nature. This study was undertaken to determine what role if any the endocrine system has in regulating the levels of INAs in Eurosta solidaginis. JH was selected because it plays an important role in insect development and diapause. Head ligation was chosen since it effectively removes the corpora allata which are responsible for JH synthesis and release. This is the first study to suggest a role for the neuroendocrine system in the regulation of INAs in Eurosta solidaginis. MATERIALS

AND

METHODS

Insect. Third-instar larvae of the tephritid fly, Eurosta solidaginis, were collected during autumn and winter just prior to experimentation. Larvae were removed from individual golden rod ball galls and maintained on moist filter paper during the various experimental regimens. Hormone application. Methoprene (Zoecon) and JH I (Sigma) were dissolved in acetone and applied topically to the larvae. Acetone-treated and untreated larvae served as controls. 1983 experiment. Larvae sampled on the third day following initial treatment were given a reapplication of hormone on the second day following initial hormone treatment; those larvae sampled on the fifth day following initial treatment were given a reapplication of hormone on the third day following initial hormone treatment. Larvae received topical applications of JH I in concentrations of 0.01 pg/Fl and either

AND

BAUST

0.1, 0.01, 0.001, or 0.0001 pg/kl for methoprene. 1984 experiment. Only one initial dose of JH I was given with no reapplications. Larvae were individually weighed and given 0.05 pg JH I per 40 mg wet weight of larva to compensate for differences in individual larval weights. Supercooling points. Supercooling points (SCP) were determined on homogenates prepared from the larvae as described by (4). Homogenates were used for supercooling points since the hemolymph was found not to contain INAs. Three to four larvae weighing approximately 150 mg were homogenized in 3.0 ml of 1.8% sucrose (w/v) to provide an approximate isotonic medium. Replicates of three capillary tubes filled with 5 ~1 of homogenate sandwiched between two layers of mineral oil (to prevent evaporation) were prepared and frozen at -45°C until used. Supercooling points were determined on a recording potentiometer by attaching a copper-constantan thermocouple to the capillary tubes containing the larval homogenates and then cooling the tubes at a constant rate of 1°C min until frozen. Supercooling points were defined as the point of inflection on the latent heat of fusion curve (11). Student’s t test for the comparison of two means was used to determine significance. The following year (1984) the experiment was repeated, this time using individual SCP of intact individual larva as well as homogenate SCPs. Individual larva were placed in separate plastic tubes tapered at one end with a 32 ga. copper-constantan thermocouple in contact with the surface of the larval cuticle. A “foam rubber” plug was inserted into the tube to hold the larva and thermocouple in position and cooled at a constant rate of l”C/min until frozen. Allatectomy. Larval allatectomy, removal of the corpora allata, was accomplished by placing a fine silk thread ligature behind the fourth segment followed by removal of the head.

COLD HARDENING

Acclimation. Acclimation temperatures of + 15 and + 5°C were chosen because previous studies have shown that + 5°C induces synthesis of the cryoprotectant sorbitol and + 15°C induces its loss (8, 1.5). Larvae from each treatment group were divided between + 15 and + 5°C and sampled on Days 1, 3, and 5. RESULTS

A. Ligation. Larvae collected from the field in 1983 and 1984 were head ligated shortly after collection. Supercooling points were determined on 5-~1 vol of homogenates prepared from pooled samples of larvae collected in 1983 and on both individual intact larvae and homogenates of larvae collected in 1984. HOMOGENATES

IN Eurostn

467

sdidaginis

The effects on supercooling points were similar for homogenates (1983) from ligated larvae acclimated to +5 and + 15°C (Figs. 1 and 2). The SCP of September larvae increased significantly 3 days following head ligation. For larvae acclimated to + 5”C, ligation resulted in an increase in the SCP from -5.7 t 1.3 to -10.8”C. Larvae acclimated to + 15°C showed a similar increase. October larvae responded significantly to ligation with elevated SCP over unligated controls within 1 day for both + 15 and + 5°C acclimated larvae. December larvae were relatively insensitive to ligation with no significant changes occurring at + 5°C over controls and with one significant change in the + 15°C group on Day 3.

(1983)

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(1984)

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IO

DECEMBER

r

NOVEMBER

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DECEMBER

DAYS DAYS FIG. 1. Comparison of the effect of allatectomy (head ligation) on supercooling point of Eur-ostu solidaginis (third-instar larvae) homogenate vs whole body. Larvae were acclimated to +S”C. Unligated larvae served as controls. Values are 2 SEM. Asterisks represent points significant from controls; all ordinate values are equal. *O.Ol < P < 0.05, **O.OOl < P < 0.001, ***P < 0.001,

468

ROJAS, HAMILTON, HOMOGENATE

AND BAUST

(1983)

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FIG. 2. Comparison of the effect of allectectomy (head ligation) on supercooling point of E. salidr~ ginis third-instar larvae homogenate vs whole body. Larvae were acclimated to + 15°C. Unligated larvae served as controls. Values are i SEM. Asterisks represent points significant from controls as defined in Fig. 1. All ordinate values are equal.

Similar results were obtained for intact ligated and unligated larvae collected the following year (1984) (Figs. 1 and 2). As in the previous year the general effect of ligation was to diminish the supercooling capacity. Also, a seasonal sensitivity to ligation was observed in the +5”C acclimation group as observed in the previous year in that November larvae showed the greatest response to ligation, while December larvae were relatively insensitive to ligation (Fig. 1). Since intact individual larvae SCP gave similar results to homogenate SCP of pooled larvae the 1983 results using homogenate SCP cannot be attributed to artifacts. B. Juvenile hormone addition to ligated allatectomized larvae. JH I was added to

ligated larvae to determine whether the effects of ligation could be partially or completely reversed. If so, this would suggest that the effects observed with ligation were due to JH deprivation and not some other factor of ligation. The 1983 experiment shows that with the addition of JH to ligated larvae there was in many cases a significant depression of the SCP over ligated acetone-treated larvae (Fig. 3). September ligated larvae given JH I had SCPs significantly lower than those of their ligated controls on Day 3 for both +5 and + 15°C acclimated larvae; however, December larvae showed no change in SCP over ligated-acetone controls. JH I addition to ligated larvae in 1984 re-

COLD HARDENING

IN Eurosfa

HOMOGENATES

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469

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(1983)

SEPTEMBER

a ligated l ligated

(1~~1 acetone) IO.01 ul JH)

5

Y--e--Y 0 DAYS

DAYS

FIG. 3. Effect of hormone (JH) addition to JH-deprived (ligated) E. sobdrcginis third-instar larvae on homogenate supercooling point. Acetone-treated larvae served as controls. Values are 2 SEM. Asterisks represent points significant from controls as defined in Fig. I. All ordinate values are equal.

sulted in the suppression of the SCP over ligated-acetone controls in only one case on Day 3 in the + 5°C acclimation group (Fig. 4). All other points from October, November, and December in both the + 5 and + 15°C groups were not significant even though the dosage of JH was higher than that in 1983. Homogenate SCPs were also utilized to determine whether the effect observed in 1983 was a function of using homogenates versus whole body SCPs (Fig. 5). C. Methoprene addition to unliguted larvae. Figure 6 shows the effect of methoprene, a JH analog, addition 3 days following initial analog treatment on supercooling capacity of larval homogenates in October unligated larvae incubated at t 5°C. Suppression of supercooling point occurred in a dose-dependent manner. The

maximal response occurred with the 0.01 ug dose. Unligated larvae given methoprene and acclimated to + 15°C had elevated supercooling points (not shown) over their controls of around 5°C for September, October, and December larvae, opposite to the effect seen for those incubated at +YC. CONCLUSION

Insect cold-hardiness is frequently associated with metabolic changes leading to accumulation of cryoprotectants, accumulation or reduction in ice-nucleating agents, or induction of thermal hysteresis factors. Environmental cues such as temperature (8), humidity, and photoperiod (5) act in some species to trigger cold-tolerance responses. How these environmental cues are translated within the organism to in-

470

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AND BAUST

Removal of the corpora allata (the endocrine gland responsible for JH synthesis, storage, and release) by head ligation results in a loss of supercooling capacity. Since JH or JH analog (methoprene) additions to ligated larvae partially suppress the elevation of the supercooling points, it is reasonable to suggest that the effect is due to removal of juvenile hormone. That this effect was not observed in 1984 should not be disconcerting since a larger dose of JH was used (at least 5 X) and applied only once. Saturation of receptors may have occurred and could explain nonrepeatability. E. solidaginis larvae from Texas have been shown to depress their supercooling point with the onset of winter by decreasing levels of ice-nucleating agents and with a

-8 -12

HOMOGENATES -16

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DAYS

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1

1

1

0

1

2

3

( 1984)

DAYS

FIG. 4. Effect of hormone (JH) addition to JH-deprived (ligated) E. solidaginis third-instar larvae on whole body supercooling point. JH dose was 0.05 *g/40 mg wet weight of larvae. Larvae were acclimated to either 5 or 15°C. Acetone-treated larvae served as controls. Values are 2 SEM. Asterisks represent points significant from controls as defined in Fig. I. All ordinate values are equal.

1

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duce changes within the cellular machinery necessary for the switching on/off of cryoprotectant or ice-nucleating synthesis is not known. Storey and Storey (15) have shown that low temperature in E. solidaginis acts on the metabolic pathways to inhibit certain enzymes resulting in the preferential shunting of carbon into cryoprotectant synthesis. This report (INAs) and that of Horwath and Duman (6) (antifreeze proteins) for the first time implicate the endocrine system (juvenile hormone) as the probable mediator between environmental cues and metabolic changes associated with cold-hardiness.

1

1

1

1

NOVEMBER

Q-T IL

I 0

1 1

1 2

1 3

I 4

DAYS

FIG. 5. Effect of hormone (JH) addition to JH-deprived (ligated) E. solidaginis third-instar larvae on supercooling point of larval homogenates. Acetonetreated larvae served as controls. JH dose was 0.05 @g JH/40 mg wet weight larvae. Values are + SEM. Asterisks represent points significant from controls as defined in Fig. 1. All ordinate values are equal.

COLD HARDENING DOSE

+5 G

RESPONSE

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(unligated~

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-5

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0.001

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DAY

3

FIG. 6. Effect of analog (methoprene) addition to unligated Eurosta solidnginis third-instar larvae on homogenate supercooling point. The homogenate supercooling point is plotted as a change from untreated (unligated) controls. (+ , elevation of supercooling point; - , depression of supercooling point over control.) Error bars represent the combined SEM of the values for the analog-treated larvae and the values of their respective controls.

IN Em~stu

471

solidqinis

E. solidaginis demonstrates a seasonal sensitivity to juvenile hormone and methoprene via perturbation of supercooling point capacity. September and October larvae are significantly more sensitive to JH than December larvae. Changes in receptor pools or in endogenous levels of JH during the development of the larvae may explain these sensitivity changes. Temperature was found to mediate the effect of methoprene on supercooling in unligated larvae. It suppresses supercooling in larvae incubated at + 5°C and elevates it in larvae incubated at + lS’C, an effect opposite to that of JH. The suppression of nucleation in ligated larvae given juvenile hormone or methoprene mimics the response of unligated larvae (+ YC). Why an elevation of supercooling points at + 15°C occurred in unligated larvae is not known. ACKNOWLEDGMENTS

buildup of cryoprotectant levels of glycerol, sorbitol, and trehalose (Baust, 1981). Juvenile hormone may either influence supercooling points by acting on INA synthesis, on cryoprotectant levels, or on both. A parallel study has shown that juvenile hormone does affect cryoprotectant levels (19). However, the changes observed in cryoprotectant levels are not sufficient to explain the changes observed in supercooling point. Dilution curves for the homogenates permit a semiquantitation of nucleator levels and reveal a higher concentration of nucleators in the ligated larvae than in the unligated larvae. It has been suggested that JH exerts its effect on target cells through binding to a specific chromosomal site(s) to induce transcription and protein synthesis (7). For example, Horwath and Duman (6) found that juvenile hormone induces antifreeze protein production in larvae of the beetle, D. canadensis. One possible mechanism through which JH could act to inhibit proteinaceous ice-nucleating agents is through induction of synthesis of a protease.

This research was supported by NSF Research Grant PCM 81-10327 to J.G.B. Special thanks are extended to Dr. G. Staal of Zoecon for supplying samples of methoprene and to Tuan-An Luu for his help in laboratory preparation of samples. REFERENCES 1. Baust, J. G. Biochemical correlates to cold hardening in insects. Cryobiology 18, 186-198 (1981). 2. Baust, J. G., Grandee, R., Condon, G., and Morissey, R. E. The diversity of overwintering strategies utilized by separate populations of gall insects. Physiol. Zoo/. 52, 572-580 (1979). 3. Baust, J. G., and Lee, R. E. Divergent mechanisms of frost hardiness in two populations of the gall fly, Eurostu solidaginis. J. Insect Physiol.

27, 485-490

(1981).

4. Baust, J. G., and Zachariassen, K. E. Seasonally active cell matrix associated ice nucleators in an insect. Cryo-Left. 4, 65-71 (1983). 5. Horwath, K. L., and Duman, J. G. Involvement of the circadian system in photoperiodic regulation of insect antifreeze proteins. J. Exp. Zoo/. 219, 233-270 (1982). 6. Horwath, K. L, and Duman, J. G. Induction of antifreeze protein production by juvenile hormone in larvae of the beetle Dendroides canudensis.

J. Comp.

Physid.

151, 233-240

(1983).

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ROJAS , HAMILTON,

7. O’Conner, J. Ecdysteroid and juvenile hormone receptors. In “Endocrinology of Insects” (Riddiford, Ed.), pp. 559-565. A. R. Liss, New York, 1983. 8. Rojas, R. R., Lee, R. E., Luu, T. A., and Baust, J. G. Temperature dependence-independence of antifreeze turnover in Eurosta solidaginis (Fitch). J. Insecr. Physiol. 29, 865-869 (1983). 9. Salt, R. W. Studies on the freezing process in insects. 1. Insect. Physioi. 27, 485-490 (1936). 10. Salt, R. W. Factors influencing nucleation in supercooled insects. Ccmod. .I. Zool, 44, 117-133 (1966). 11. Salt, R. W. Effect of cooling rate on the freezing temperatures of supercooled insects. Cunad. J. Zool. 44, 655-659 (1966). 12. Salt, R. W. Location and quantitative aspects of ice nucleators in insects. Can&. J. Zoo/. 44, 947-952 (1968). 13. Somme, L. Nucleating agents in the haemolymph of third instar larvae of Eurosta soliduginis (Fitch) (Dipt. Tephritidae). Now. J. Entomol. 25, 187-188 (1978).

AND BAUST 14. Somme, L. Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. 73A, 519-543 (1982). 15. Storey, K. B., and Storey, J. M. Biochemical strategies of overwintering in the gall fly larva, ELUOSQ solidaginis: Effect of low temperature acclimation on the intermediary metabolism. J. Comp. fhysiol. 144, 191-199 (1981). 16. Zachariassen, K. E. Nucleating agents in coldhardy insects. Comp. Biochem. Physiol. 73A, 557-562 (1982). 17. Zachariassen, K. E., Baust, J. G., and Lee, R. E. A method for quantitative determination of ice nucleating agents in insect hemolymph. Cryobiology 19, 180-184 (1982). 18. Zachariassen, K. E., and Hammel, H. T. Nucleating agents in the haemolymph of insects tolerant to freezing. Nature (London) 262, 285-287 (1976). 19. Hamilton, M. D., Rojas, R. R., and Baust, J. G. Juvenile hormone: modulation of cryoprotectant synthesis in Eurosta solidaginis by a component of the endocrine system. J. Insect Physiol32, 971-979 (1986).