Juvenile hormone: Modulation of cryoprotectant synthesis in Eurosta solidaginis by a component of the endocrine system

0022-1910/86 53.00 + 0.00

J. Insect Physiol. Vol. 32, No. 11, PP. 971-919, 1986 Rintcd in Great Britain

Pergamon Journals Ltd

JUVENILE HORMONE: MODULATION OF CRYOPROTECTANT SYNTHESIS IN EUROSTA SOLIDAGINIS BY A COMPONENT OF THE ENDOCRINE SYSTEM M. D. HAMILTON, R. R. Ror~s and J. G. BAUST* Institute of Low Temperature Biology, University of Houston-University Park, Houston, TX 77004, U.S.A. (Received 20 February 1986; revised 3 March 1986)

Almtract-The role of juvenile hormone as a possible modulator of insect cold-hardiness has been investigated in the gall fly larvae of Eurosta solidaginis. The 3rd~instar larva is freezing tolerant and survives the rigours of winter by producing carbohydrate cryoprotective agents (i.e. glycerol and sorbitol). Since cold-hardening frequently occurs in conjunction with developmental processes under endocrine control, a study of these possible interrelationships was made. Third-instar larvae collected during autumn were allatectomized by ligation of the head. Experimental larvae received topical applications of synthetic juvenile hormone, methoprene, a potent juvenile hormone analogue, or precocene II, an anti-juvenoid. Following treatment, all larvae were then subjected to one of two temperature acclimation protocols at 5 or 15°C for up to 5 days. Ligation resulted in a 20% decrease in glycerol and up to a 40% increase in sorbitol levels vs controls following 5°C exposure. At 15”C, deprivation yielded a 13% decrease in glycerol and up to a 47% increase in sorbitol. Ligation resulted in decreased glycerol levels independent of temperature especially in early autumn. Cryoprotectant synthesis/accumulation was responsive to juvenile hormone replacement with peak sensitivity observed in October. Ligated larvae were generally insensitive to juvenile hormone replacement in September and December. These responses were temperature dependent; polyol accumulation was reduced by 50% at +5”C and enhanced by 22% at + 15°C (2 and 8 pg glycerol/mg larvae, respectively). This study provides the first evidence suggesting an association between juvenile hormone and regulation of cryoprotectant accumulation in insects. Key Word Index: Juvenile hormone, cold hardiness, antifreeze, cryoprotectants,

INTRODUCTION

Terrestrial arthropods resident to polar and temperate regions possess the ability to withstand low temperatures and survive the rigours of winter. Since the pioneering studies of Salt (1957, 1961), research on the strategies of cold tolerance in insects has intensified and several categories of adaptive mechanisms have been identified. Overwintering insects generally accumulate low-molecular-weight polyhydroxy alcohols (i.e. glycerol, sorbitol, etc.) and sugars (Salt, 1957; Somme, 1964; Baust and Miller, 1970, 1972; Morrissey and Baust, 1976; Baust and Edwards, 1979). Overwintering insects are commonly described as freeze intolerant or tolerant. If a species is intolerant to freezing, its supercooling capacity is usually extended to temperatures below those encountered during winter, allowing for the maintenance of body fluids in the liquid state. The extension of supercooling capacity due to antifreeze accumulation exceeds the corresponding equilibrium freezing point depression of body fluids by a factor of 1.5-2.5 (Franks, 1981). Freezing-susceptible species also demonstrate an ability to modify ice nucleator pools obtained either from exogenous sources or by de nova synthesis (Baust, 1982, Baust and Zachariassen, 1983; Baust et al., 1979; Somme, 1982). *To whom correspondence should he addressed.

freezing tolerance

Recent studies have focused on the identification of external parameters responsible for the initiation (triggering) and maintenance of cold-hardiness. Duman (1977) has shown that photoperiod is a stimulus to the triggering of antifreeze protein production in Mercantha contracta. Humidity (Young and Block, 1980) may also play a role in the introduction of cold-hardening, as well as ingested substances (Somme and Conradi-Larsen, 1977). Low temperature, however, has been the primary cue for triggering a diverse set of insect hardening strategies (Baust and Miller, 1970, 1972; Ziegler and Wyatt, 1975; Ring, 1977; Baust er al., 1979; Storey et al., 1981; Rojas et al., 1983). Eurosta soiidaginis, a dipteran, inhabits the goldenrod stem gall and over-winters in the 3rd~larval instar. This species utilizes an adaptive strategy that relies on the accumulation of cryoprotective polyols beginning in September and reaching a maximal level in December. Also, there is a concomitant decrease in glycogen stores in the fat body as metabolic pathways shift to facilitate the hardening process (Morrissey and Baust, 1976; Storey et al., 1981). Environmental temperature regulates, in part, the production of polyols (Baust and Lee, 1982). Sorbitol synthesis in particular (Rojas et al., 1983) has been shown to be temperature dependent. The triggering event responsible for glycerol synthesis, however, is unclear (Baust, 1983). Storey and Storey (1982) propose that

971

972

M. D. HAMILTONet al

glycerol production in E. soliduginis is temperature dependent in northern populations, while the results of Rojas et al. (1983) demonstrate that glycerol synthesis is temperature independent in southern populations and probably more directly related to cellular water perturbation (Baust and Lee, 1982). In addition, the initial accumulation of glycerol in early autumn is anticipatory, since levels rise before the first cold exposures (Baust and Morrissey, 1977; Baust, 1985). Relatively little data are available relating to the internal mechanisms responsible for in situ triggering and regulation of polyol synthesis. Storey (1982, 1983) has shown that temperature has a direct influence on a key glycolytic regulating enzyme, phosphofructokinase, and the routing of carbon through the glycolytic pathway. Tsumuki et al. (1987) have also reported on temperature control of glycolytic routing of carbon in E. solidaginis. However, there is a high probability that physiological regulation of cryoprotectant levels involves coordination above the level of modulation of one or a few enzymes. As the autumn progresses, the hardening larvae may either sustain or arrest developmental processes regulated by a temporal sequence of endocrinological events. Hormones essential to these developmental processes are known to direct a number of metabolic activities in insects unrelated to cold hardening (see reviews by Wyatt, 1972; Sekeris, 1974; Steele, 1976). Several hormones are known to affect carbohydrate metabolism (Steele, 1963; Liu, 1973). In particular, juvenile hormone regulates haemolymph carbohydrate levels through the control of glycogen metabolism (Butterworth and Bodenstien, 1969; Ziegler and Wyatt, 1975). Horwath and Duman (1983) provided the first report on the probable regulatory influence of this hormone on the production of thermal hysteresis proteins in Dendroides canaaknsis. Evidence directed toward endocrine mediation of carbohydrate (polyol) turnover during cold hardening is wanting. Preliminary experiments in our laboratory suggest the existence of a blood-borne factor which influences the accumulation of polyols during pre-winter hardening. This study, which utilized the techniques of hormone elimination (chemical and surgical allatectomy) and replacement therapy, provides the first data supporting a hypothesis of neuroendocrine control of low molecularweight antifreeze/cryoprotectant production during the cold-hardening process. MATERIALS

AND METHODS

Experimental animals

Goldenrod stem galls containing 3rd-instar larvae of Eurosta solidaginis (Diptera: Tephritidae) were collected along the coastal plain of southeast Texas. Single collections were made each month during September, October, and early December. On the day following collection, the larvae were extracted from the gall, placed in sterile Petri plates containing moistened filter paper and maintained at near ambient temperatures (1-2 days). Ablation of the corpora allata

Experimental

larvae were allatectomized

by liga-

tion of the head. Upon ligature of the body between the fourth and fifth segments using a very thin silk thread, the head was excised. This procedure ensured the elimination of the ring gland and the brain. The remaining body of the larvae was effectively deprived of any signals originating from the lateral or medial neurosecretory glands, corpora cardacia, prothoracic glands and corpora allata. All control information from the head region was essentially disrupted. Such an extreme method may appear unnecessary in light of other procedures which eliminate the corpora allata specifically. A number of problems exist, however, with employment of these alternate procedures in the 3rdinstar larvae of Eurosta solidaginis. First, the complex interactions of the endocrine system and the nervous system make it difficult to manipulate only one component of a heretofore unstudied system without destabilizing all other associated components. Surgical removal of the corpora allata alone is impractical because of size limitations and since it is an integral part of the ring gland. Experiments employing an antijuvenile hormone compound have also been included in this work although the effectiveness of using precocene to chemically and selectively destroy the corpora aflata is arguable. Some antijuvenile activity was achieved in Drosophila larvae (Lander and Happ, 1980; Wilson et al., 1983), yet definitive effects on the corpora allata have not been substantiated (Kelly and Fuchs, 1978; Bowers, 1982). Precocene is an antijuvenile hormone found in natural systems, mainly plants. It is specifically cytotoxic to the insect corpora allata, site of juvenile hormone synthesis and release (Bowers, 1983). Rapid destruction of corpus allatum tissue results in the reduction or termination of juvenile hormone synthesis. Responsible for several types of biological activity, precocene has been an effective tool in the exploration of insect physiological and behavioural development now understood to be regulated by juvenile hqrmone (i.e. precocious metamorphosis, antigonadotropic and antipheromonal activity, diapause regulation and caste morph regulation). Assuming that it is effective in dipterans and that the absence of juvenile hormone would influence cryoprotectant polyol synthesis (carbohydrate metabolism), precocene offered a less invasive alternative to ligation (surgical extirpation) of the corpora allata. Similar responses between these two methods would help demonstrate that the juvenile hormone titre does indeed influence polyol production during the cold-hardening process. (Note: measurements of endogenous levels are not yet possible in dipterans). The ligation procedure is therefore designed to remove all signals/messengers and replace only one (juvenile hormone and its analogue). The effect of replacement on cryoprotectant accumulation can indicate an exclusive involvement of juvenile hormone on the coldhardening process. No significant water loss was observed (body weight) following 1, 3 and 5 days of acclimation. Hormone applications

Juvenile hormone I (Sigma), ZR-515 (methoprene), a synthetic juvenile hormone (Zoecon), and precocene (Sigma) were dissolved in acetone and ap-

973

Modulation of cryoprotectant synthesis plied, directly to the thin cuticle by precisely delivered drops from a microsyringe (1 $/larvae). Doses given to the ligated larvae were 0.01 pg/pI juvenile hermone 1 or 0.1, 0.01, 0.001, 0.0001 pg/@l of methoprene. The dose-response optimum for precocene was 0.5 fig/ml/40 mg larval wet weight (Note: seasonal water content is invariate in this species). The treated larvae were then placed in Petri plates with moistened filter paper and incubated at f5”C f 0.5”C or + 15°C f 0.5”C for periods of 1, 3 and 5 days. A reapplication of hormone or analogue was given on day 2 for those larvae undergoing a 3-day exposure or on day 3 for larvae exposed 5 days. Controls for each day and temperature included (1) unligated, untreated larvae, (2) ligated, untreated larvae, and (3) ligated larvae receiving 1 pl of the acetone carrier. All unligated larvae were fasted for the duration of the experiment. Following the incubation period, the larvae were weighed and quick frozen to -40°C.

l

/

012345

Extraction of polyols

+ 15’C

+ 5-c 2140 F

.

012345

Days

Each sample to be analyzed (3-4 larvae per sample) was homogenized in 2 ml 1.8% sucrose solution, vortexed in 2 ml of 95% ethanol and placed in a hot water bath for 5 min. The homogenate was cooled and centrifuged at 3,000 rpm for 30 min until a clear supematant was obtained. The supematant was collected and the pellet was washed with 2ml of 95% ethanol, vortexed and centrifuged. The supernatants were combined and evaporated to dryness. The evaporated residue was resuspended in 2.0ml deionized water and partitioned against an equal volume of chloroform-methanol (2: 1, v/v). The resuspended and partitioned residue was thoroughly vortexed, centrifuged at 10,000 rpm for 30 min, and the aqueous phase removed. The remaining organic layer was washed with 2 ml water, centrifuged, and the aqueous phases combined and evaporated to dryness. Each extract was resuspended in 0.5 ml of distilled water and filtered (0.4~0 for HPLC analysis. Carbohydrate and polyol types and levels were determined by high pressure liquid chromatography (Baust et al., 1983). A sample weight of at least 130mg was necessary for an accurate quantifiable assay of polyol content using HPLC. It was essential to pool 3-4 larvae from the same condition to obtain a combined weight of 130mg. Each experimental condition utilized 12 larvae and a total of 576 larvae were utilized each month. Effects described as being significantly different from control values were determined by the Student’s t-test (P < 0.05). RESULTS Experiments describing the hormonal influence on cryoprotective polyol synthesis have not previously been reported. The design of these experiments was to screen for effects on polyol accumulation in E. solidaginis larvae by neuro-endocrine deprivation and by specific replacement of juvenile hormone. Parameters inherent to the cold-hardening process, i.e. temperature, duration of exposure and season, were varied to determine their interaction with endocrine manipulation. Therefore, effects on the two major antifreeze/cryoprotective agents (glycerol and sor-

Glycerol

Days

Fig. 1. Seasonal response of glycerol (O), and sorbitol (A) following exposure to + 5°C and + 15°C for I,3 and 5 days. Error bars represent standard error. Levels measured in pg polyol/mg (wet weight) larvae.

bitol) following treatment and acclimation to +5”C or + 15°C are described in the following order: glycerol synthesis in untreated larvae (control) and ligation (juvenile hormone elimination), acetone treated (ligated control) larvae, juvenile hormone replacement (ligated larvae) and methoprene (ligated larvae). Sorbitol synthesis is described in the same order as glycerol synthesis. Finally, effects induced by precocene on both glycerol and sorbitol are described. Glycerol synthesis: control us ligation Following the first day of exposure to +5”C (September), unligated, untreated larvae demonstrated an elevation in glycerol levels vs controls (Fig. 1). Levels reached a maximum by the third day of exposure. This pattern persisted for these control larvae in October and December (i.e. a rise above time zero samples following 1 day of incubation, peak levels by day 3 and a decrease by the fifth day). There is, however, an overall seasonal augmentation of glycerol production beginning with the lowest mean levels in September to the highest in December. Intact larvae acclimated at 15°C had increased glycerol stores over the 5-day period (Fig. 1). For each month, polyol measurements of untreated larvae exposed to this temperature yielded higher glycerol content vs time zero samples after 1 day and remained high through the 5-day acclimation interval. Glycerol content did not decrease from time zero levels to the extent that sorbitol had following 1 day of acclimation at either temperature in December (Fig. 1). Head ligation, removal of the ring gland containing the corpora allata, results in the impairment of juvenile hormone synthesis and release and presumably results in a rapid reduction in the juvenile hormone titre. A significant reduction (P < 0.05) in

914

M. D. HAMILTONet al. + 15’C

G E

6 r

October

L

L-

I

Days

1

2

3

4

Days

Days

Fig. 2. Seasonal response of glycerol accumulation in Euroand acclimation to + 5°C and + 15°C for 1, 3 and 5 days. The unligated untreated controls are represented by closed circles (a). Error bars represent standard error.

sta sofiduginis following ligation (0)

the glycerol synthetic capacity occurred following head ligation for all September samples independent of temperature (Fig. 2). Comparatively lower levels of glycerol were also seen in October and December samples. In October reduced levels were measured following 1 day at +S’C and became significantly reduced by the fifth day. Glycerol levels measured in December ligated samples were lower than the controls following 1 day of acclimation at 5°C and significantly lower after 3 days. At the 15°C acclimation in December ligation resulted in a significant decrease in glycerol levels following 3 and 5 days. An average 2~g/mg weight decrease in glycerol pools was calculated for ligated samples (Fig. 2).

c-

012345

012345

5

A --8-----e

9

y

of-, , , , , , 0

I

c

December

11 y

I

l ;sp 1

13 r

r

I

Days

Fig. 3. Response of glycerol accumulation following ligation and topical application of 1~1 acetone and acclimation for 1, 3 and 5 days to +5”C and + 15°C. Acetone-treated ligated larvae are represented by closed squares (m) and the ligated untreated control by open boxes (0). Error bars represent standard error of the samples.

Exposure to 15°C led to significantly (P < 0.05) higher glycerol levels in hormone treated larvae after 1 and 5 days. In October samples a dichotomy in the character of response occurred in hormone-treated larvae between the two acclimation temperatures (Fig. 4). Glycerol levels of hormone-treated larvae +5’C 6

+ 15*c

r

September

4

Glycerol synthesis: acetone effects

Since juvenile hormone was dissolved in an acetone carrier, an analysis of acetone’s effect on ligated larvae was made (Fig. 3). Analysis of the data revealed that the acetone treatment did not result in a significant alteration of glycerol levels compared to untreated controls at either the 5 or 15°C acclimation in September, October or December (P < 0.05, t-test). Glycerol synthesis: analogue potentiation and juvenile hormone replacement

Additions of juvenile hormone of its analogue, methoprene, were made in an attempt to restore only one of several hormones and neurosecretory products that were eliminated by ligation. Any effect would then suggest an association between juvenile hormone and cryoprotectant synthesis. Application of juvenile hormone (0.01 pg/larvae) to ligated larvae resulted in an increase in glycerol accumulation over ligated acetone controls following acclimation at 5 or 15°C in September (Fig. 4).

016 E 26 24 5k

2

t,

g0 0

I 15 r

I

I

I

I

,

December

I

,

,

,

,

I

r

13 11 9 7 E 0*

I I 012345

I Days

I

I

I

LI012345 Days

Fig. 4. Seasonal response of glycerol accumulation following ligation, addition of 0.01~1 juvenile hormone/larvae and subsequent acclimation to +5”C or + 15°C for 1, 3 and 5 days. Honnone-treated larva are represented by closed triangles (A) and acetone-treated controls by closed boxes (m). Error bars represent standard error of the sample.

975

Modulation of cryoprotectant synthesis t15.C

+ 5-c

October F

t,

*&$4 ) I, I,

VI 6 4 2 0

l ,(” A

I

1

1

012345

’

.

Ligation resulted in levels of sorbitol that were either similar or slightly lower than untreated unligated larvae for the September acclimation periods (Fig. 5). In contrast, ligation resulted in a stimulation of sorbitol synthesis during October at both the 5 and 15°C regimes. The effects of ligation in the December samples (increased sorbitol levels) were seen after 5 days of acclimation at 5”C, while at 15°C ligation resulted in significantly increased sorbitol content (P < 0.05) after 1 and 5 days of acclimation. Sorbitol synthesis: acetone effects

Since acetone was used as the vehicle for topical application of juvenile hormone, its effect on ligated alone was tested by applying 1 /.Qlarvae and subsequent acclimation to 5 and 15°C. Analysis of the resulting sorbitol levels revealed that acetone did not have a significant effect on sorbitol accumulation (Fig. 6).

1 1 012345

Days

Sorbitol synthesis: juvenile hormone replacement Days

Fig. 5. Seasonal response of sorbitol accumulation in EuroSIP solidaginis following ligation (0) and acclimation at + 5°C or + 15°C for 1, 3 and 5 days. Unligated untreated controls are represented by closed circles (a). Error bar represents standard error of the samples.

acclimated to 5°C were reduced following 1 day and significantly reduced following 3 days of acclimation, while exposure to the 15°C regime enhanced glycerol synthesis following 1 day and significantly so following 3 days of acclimation. However, beyond 3 days of acclimation these effects were reversed (Fig. 4). A significant increase in glycerol levels vs controls was also measured for 3 days exposure to 5°C in December.

Addition of juvenile hormone 1 to ligated larvae induced a variety of changes in sorbitvl production following 5 and 15°C exposures (Fig. 7). In September, hormone-treated ligated larvae had increased sorbitol levels vs acetone controls following a 5-day acclimation at either temperature, yet only the 15°C condition was significantly different, 12.Opg sorbitol/mg larvae (P < 0.01). Replacement of exogenous juvenile hormone to ligated Iarvae in October resulted in inhibited sorbitol synthesis after 1 day and significantly after 3 days acclimation to 5°C. However, increased levels of sorbitol were observed following 5 days of acclimation. Juvenile hormone additions resulted in enhanced sorbitol levels, significantly greater after 3 days, in the warm aceli-

Methoprene

+ 5*c

+15’C

The effort to establish a dose-response curve for methoprene was unsuccessful. Levels of polyols following treatment and acclimation temperature exposures of any duration over the 3 months were inconsistent. It would appear that methoprene is not a suitable analogue to juvenile hormone in this species.

September

F

Sorbitol synthesis: control us ligation

Untreated, unligated larvae exposed to 5 or 15°C served as controls (Fig. 1). September larvae after 1 day of acclimation contained slightly higher levels of sorbitol vs time zero at either temperature. Except for the 5-day 5°C acclimation, the sorbitol levels plateaued at approximately the levels measured for 1 day acclimation. These larvae acclimated at 5°C in Cktober had increased sorbitol levels following 3 days of exposure while the 15°C acclimation maintained low levels of sorbitol over the 5 day period (Fig. 1). In December, untreated unligated larvae had reduced sorbitol levels following 1 day acclimation at either temperature. The reduction is perhaps due to colder ambient temperatures for that time of season compared to the laboratory acclimation temperatures. A minor increase in sorbitol occurred during 5°C acclimation after 3 days, but, at 15°C levels remained low.

Dscember

I_ B-9

OI 0

12

34 Days

5

0

12

q

345

DCVS

Fig. 6. Response of sorbitol accumulation following ligation, topical application of 1~1 acetone and acclimation to + 5°C or + 15°C for 1,3 and 5 days. Acetone-treated ligated larvae are represented by closed boxes (B) and the ligated untteated larvae by open boxes (0). Error bars represent standard error of the samples.

M. D.

HAMILTONef al.

contribute to an effect on polyol synthesis. Statistically, acetone addition did not significantly alter ~01~01 levels vs controls (2 out of 24 experimental situations), although they often had the largest variation within a sample group. Precocene treatments compared to the untreated controls were only significantly different (P < 0.05) in 3 out of 24 experimental situations for glycerol and 1 out of 24 for sorbitol. When compared to the acetone controls (Tables 1 and 2) precocene treatments were different in only l/24 cases for glycerol and 3124 cases for sorbitol. Finally, precocene treatments should have produced the same effect on polyol accumulation as ligation, assuming that precocene did destroy the corpora allata. However, statistical analysis revealed that precocene in 5 out of 24 experimental situations for glycerol and 4 out 24 for sorbitol differed significantly from ligation (Tables 1 and 2). Interestingly, this last comparison, precocene treated verses ligated, had the greatest number of situations that differed when contrasted with the others. Thus, these experiments indicate that precocene was not a substantial factor in the influence of polyol accumulation in E. soiidaginis.

Seplember

12 6 4 0 G

t

December

\

I

I

I

I

I

I

012345

Days

Days

Fig. 7. Seasonal response of sorbitol accumulation following ligation, addition of 0.01~1 juvenile hormone/larvae and subsequent acclimation to +YC or f15”C for 1, 3 and 5 days. Hormone-treated larva are represented by closed triangles (A) and acetone-treated controls by closed boxes (W). Error bars represent standard error of the samples.

DISCUSSION

Ligation between the third and fourth anterior segments of Eurosta 3rd-instar larvae effectively eliminates all neural and hormonal signals to the posterior larval segments. As observed in Fig. 2, ligation resulted in a significant (P < 0.05) decrease in glycerol content following 1,3 and 5 day 5°C acclimations in September collections. This trend, which occurred again following 5°C acclimation in October and December (Fig. 1) suggests that a factor(s) eliminated by ligation may be required for glycerol accumulation. The response of glycerol to ligation is independent of temperature since warm acclimation (15°C) was characterized by a consistent reduction in glycerol levels following ligation (Fig. 2). Note that in the 15°C acclimation temperature the difference in glycerol levels between ligated and unligated larvae increases over time. Therefore, the full effect of ligation

result contrasted with the predicted non-stimulatory effect observed in previous warm acclimations. Maximal levels were 12.0 /4g sorbitol/mg larvae after 3 days at 15°C. Note that this pattern of response is identical to that described for glycerol accumulation in October. December ligated larvae treated with juvenile hormone, though not statistically supported, had slightly higher sorbitol levels independent of temperature. mation.

This

Precocene treatments The effects produced by precocene were compared to 3 other treatments: the untreated unligated control, the acetone control, and ligation alone. Acetone treatment of unligated larvae was also compared to untreated controls to ensure that acetone did not

Table 1. Seasonal

values of sorbitol

Precocene Month

October

November

December

Day

+ 5°C

@g/mg

larva) following

treatment

and acclimation

Untreated control

vs +lsOc

+5”c

Acetone +15”c

+ 5°C

Ligation +15”c

1 2 3 4

7.0 8.5 8.5 9.1

f f f *

1.1 0.1 1.1 0.7

3.4 f 2.1 1.3 f 0.3 2.1 f 1.0 1.3kO.5

7.0 8.7 5.4 ‘4.6

f f f f

0.3 2.0 0.8 0.3

8.5 4.1 2.2 4.3

f f f +

0.4 1.2 0.1 1.6

10.4 5.2 7.4 8.8

f f f f

0.9 0.4 0.3 0.7

1 2 3 4

3.9 4.9 7.2 12.9

* k f +

0.9 0.6 1.0 1.4

1.4kO.7 2.9 f 0.4 2.1 kO.5 3.7 * 1.3

4.2 8.7 7.8 14.2

f 0.9 f 2.3 kO.1 i 0.7

3.3 3.7 3.6 6.1

* 1.1 * 1.1 f 0.2 +2.3

3.2 8.1 9.8 12.4

f + + i

0.1. 1.4 1.0 3.3

1.4 * 1.9 f 14.1 * OS.4 +

1

4.0 6.6 9.4 9.7

* * f f

0.4 1.2 0.2 1.0

4.7 f 3.4 f 4.7 * 5.4*

* 5 k *

5.6 f 1.1 2.8kO.l 3.3 * 0.6 5.8 f 1.0

4.3 4.6 9.0 10.6

k f f f

0.6 1.O 0.4 0.7

3.0 3.4 5.2 4.1

2 3 4

Precwme values were compared with an asterisk.

1.3 0.5 1.1 1.2

to untreated

4.1 5.2 8.3 9.6

0.7 0.6 0.6 1.3

to 5°C or 15°C

control, acetone control and ligation values. Significant

+ 15°C

8.5 9.6 13.0 12.6

+ f + f

0.7 1.0 1.5 2.5

8.7 1.8 1.5 3.8

f 1.9 +O.l +0.9 f 0.8

0.2 0.6 0.2 1.0

3.0 l10.5 14.2 18.9

* * f f

0.4 1.0 3.0 3.0

1.1 ‘5.6 ‘9.9 ‘10.3

f0.2 k 0.7 * 2.5 f 1.4

0.4 0.5 0.4 0.7

4.9 6.7 9.4 11.1

* f + f

0.3 1.5 0.8 1.2

4.4 3.1 5.4 2.8

It. 0.8 *0.2 f 0.7 f 0.3

8.0 f 0.6 1.2 f0.8 2.4 k 0.4 2.6 * 1.3

* * f f

+ 5°C

differences (r-test, P < 0.05) are marked

917

Modulation of cryoprotectant synthesis Table 2. Seasonal values of glycerol bg/rnr Prccocene vs Month

October

Dav,

+5”c

1 2 3 4

5.0 It 0.6 8.3 f 0.9 6.0 + 0.5 6.1 f0.6

2 3 4

10.3 * 7.3 f 7.9 f 8.3 +

1 2 3 4

13.1 +_2.0 13.2 k 0.9 10.5 * 0.9 12.8 f 1.7

1

November

December

1.7 I.8 1.o 0.4

larva) following treatment and acclimation to 5 or 1s”C

Untreated control

+lsOc 4.6 f 6.8 + 6.7 k 7.3 *

0.2 0.5 0.1 0.5

5.5 * 0.4 5.9 & 0.8 5.6 f 0.5 6.4 + 0.6

8.2 * 7.6 + 16.0 f 9.5 f

0.4 0.6 0.7 0.4

8.0 & 0.3 7.6 + 0.3 ‘10.6f 1.1 7.6 k I .O

11.9 +0.3 11.750.6 11.2* 1.1 14.7 f 1.6

9.2 +_1.2 12.0 f 0.7 11.2kO.7 11.5*0.7

Acetone +lsOc

+5-z

+5”c

7.9 k 4.5 k 8.4 k 8.7 +

0.8 0.8 0.5 1.1

4.0 * 7.0 * 5.8 5 8.0 k

7.9 k 7.4 * ‘10.3 f 9.2 +

0.6 0.3 0.7 0.8

6.2 _+0.3 6.6 + 0.9 7.7 * 0.2 9.0 k 0.6

11.4+_ 1.2 12.1 + 0.8 12.0 + 0.7 12.7kO.9

10.9 f 11.6 f 12.3 k 10.3 f

0.2 0.6 0.3 0.9

0.5 0.5 0.6 0.5

Ligation +lsOc

5.9 f 0.4 7.1 f 0.6 8.5 f 0.8 8.0+0.1

+ 5°C 4.7 f l4.4 f 6.5 f 5.2 f

0.7 0.3 0.4 1.1

+15”c l5.5 * l4.1 * 4.9 f 6.4 f

0.1 0.8 1.7 0.6

0.5 I .4 0.3 I .O

7.7 * 0.4 6.1 f0.7 8.5 + 1.5 5.2 f 1.1

l6.3 i_ 0.2 6.2 f 0.3 *IO.8 + 0.8 6.4 f 0.6

12.7 +_1.1 15.2 f 2.0 12.9 + 0.5 12.4 & 0.6

10.6 +_1.1 Il.4 f 0.7 10.2 f 0.8 10.4 & 0.3

11.8 +_O.l 9.0 f 1.0 9.7 f 0.6 9.3 2 0.8

8.0 * 8.9 f 99.5 * 8.0 k

Precocene values were compared to untreated control, acetone control and ligation values. Significant differences (r-test, P -z 0.05) are marked with an asterisk.

(deprivation of the critical factor) is maximized over time. In most cases, glycerol content of ligated larvae remained at relatively low unchanged levels over the May period (Fig. 5). In this case, ligation presumably shuts down the synthesis machinery completely. Although a seasonal profile of hormone titres is not available for Eurostu solidaginis, a typical pattern of juvenile hormone synthesis, release and breakdown for most holometabolous insect species can be useful in the interpretation of the results. Shortly after the last moult, penultimate-stage larvae have steadily decreasing concentrations of juvenile hormone. The absence of endogenous juvenile hormone allows target tissues to be reprogrammed for the expression of the pupal phenotype in response to the first burst of ecdysone (see review by Riddiford, 1980). Since E. sofiaizginis moults to its final larval instar in early autumn (September) the juvenile hormone titre in E. solidaginis is likely to be highest in September, gradually lowering to minimal levels in December and January. Therefore, juvenile hormone, if important to the modulation of glycerol accumulation, would be most critical if eliminated in Sep tember. This is supported by the results of ligation in September, where the levels of glycerol for ligated larvae were all significantly decreased from unligated controls. According to Wigglesworth (1963) and Riddiford (1976), if target cells are to maintain a juvenilizing programme, juvenile hormone must be present at physiological concentrations. Otherwise the target tissues, beyond a critical period in the last instar, will no longer respond to juvenile hormone. There is then a period of sensitivity to the hormone in the last instar which, if exposed to the hormone, will keep the genes responsible for the phenotypic expression of a pupal morphology inactive (Riddiford, 1981). A period of sensitivity of ligated larvae to juvenile hormone replacement was observed in the response of glycerol (Fig. 7). Hormone-treated ligated larvae in October are characterized by significant changes (compared to controls) in glycerol levels over time and extreme differences in the responses between temperatures. The dichotomy in glycerol levels between temperatures occurred as a significant in-

hibition of glycerol synthesis in the 5°C acclimation for up to 3 days, while at 15°C glycerol accumulation in treated larvae was significantly enhanced up to 3 days (Fig. 7). Following 5-day acclimation, however, the pattern was drastically altered. The significant changes measured for S-day acclimated larvae can be attributed to the activity of juvenile hormone esterases that, within 5 days, have hydrolyzed the exogenous juvenile hormone and subsequently no longer affect hormone-treated ligated larval. Work on esterases that hydrolyze juvenile hormone to inactive juvenile hormone-acid or-diol have shown that the hormone in the haemolymph remains in the active form for 14 days (Bean et al., 1982; Lesserman and Herman, 1984). The difference in the response between temperatures suggests a temperaturedependent association between juvenile hormone and glycerol production. September 15°C samples treated with the hormone were also significantly higher in glycerol content than acetone controls (day 1 and 5). Responses of juvenile hormone-treated ligated larvae sampled in September and especially December were dissimilar to responses observed in October and are apparently due to a reduced sensitivity to the hormone. Periodic sensitivity to juvenile hormone has been reported in other species (Riddiford, 1980; Nijhout, 1983; Shenal, 1983). Sorbitol, a second major cryoprotectant in Eurosta solidaginis, is already known to be controlled by temperature (Rojas et al., 1983; Baust and Lee, 1981). The enzyme phosphofructokinase functions as a temperature dependent on/off switch. Cold acclimation diverts carbon in the glycolytic pathway to the pentose phosphate shunt by lowering the activity of phosphofructokinase (Storey, 1982; Storey and Storey, 1982). The effect of ligation illustrated in Fig. 2 is characterized by slight increases in sorbitol over ligated controls and, unlike glycerol, the changes resulting from ligation are more pronounced in October and December. The increases are not large, but these results suggest that deprivation of some factor(s) causes an increase in sorbitol accumulation. It must be kept in mind that the physiological stress of ligation (the distribution of the larva’s anterior region) may increase sorbitol synthesis regardless of hormone deprivation.

978

M. D. HAMILTON etal.

Evidence for the influence of juvenile hormone is supported by the juvenile hormone replacement experiments. Recorded levels of sorbitol following hormone addition and acclimation at either temperature reveal that a pattern of sensitivity occurred similar to the responses of glycerol under the same conditions (Fig. 7). With the exception of the 5-day 15°C sample, September larvae showed little change in sorbitol content when given juvenile hormone. This inactivity was also observed in the December experiments. October samples responded to juvenile hormone replacement with alterations of the greatest magnitude versus controls. As seen with glycerol, exogenous juvenile hormone inhibited sorbitol production significantly up to 3 days in the 5°C regime. During this period of inhibition the predicted cold temperature induction of sorbitol production was overridden by the hormone. By the fifth day of acclimation the influence of juvenile hormone had ceased, presumably due to its inactivation by esterases. In the 15°C regime the hormone-treated ligated larvae had significantly increased sorbitol pools following 3 days of acclimation. Normally, sorbitol pools in E. solidaginis remain low at warmer acclimation (Baust, 1976) but the increase indicates an influence of the hormone. By the fifth day, treated larvae contained significantly reduced levels of sorbitol, which indicates a loss of activity and/or degradation of the exogenous hormone. Experiments using precocene as an alternate method of eliminating endogenous juvenile hormone have been included here, even though they were conducted the following autumn. Since precocene is used for its antijuvenile hormone effects in some insect species, it was important to demonstrate whether or not precocene could be used instead of ligation (which is a much less specific and often lethal procedure). The resulting polyol levels from precocene treatments compared to the levels resulting from ligation had dissimilar patterns. Statistical comparisons revealed that polyol levels of larvae treated with precocene did not significantly differ from acetone controls or untreated larvae. In summary, these experiments indicate an association between the juvenile hormone and cryoprotectant accumulation. Ligation results in a decrease in glycerol levels and a minor increase in sorbitol levels. Replacement of juvenile hormone, one of many factors from the anterior region removed by ligation, revealed that Eurosta larvae have a seasonal sensitivity to hormone replacement. Peak responses occur in October while September and December were relatively insensitive. Juvenile hormone replacement was characterized by significant inhibition of cryoprotectants following acclimation to 5°C while after a 15°C acclimation polyol levels were significantly enhanced. The inhibition at 5°C and the enhancement at 15°C were not observed beyond a 3-day acclimation, presumably due to degradation of the hormone by juvenile esterases. The dichotomous response to the replacement of juvenile hormone between the two temperature regimes (5 and lS°C) suggests that the larval target cells have a temperature-dependent response with respect to polyol accumulation. Methoprene did not have a consistent nor significant effect on polyol accumulation in

3rd-instar larvae. Precocene did not mimic the response for ligation. Acknowledgements-This research was supported by NSF research grant PCM al-10327 to JGB. Special thanks to Gerald Staal of Zoecon for supplying samples of methoprene and to Tuan-Anh Luu for his help in laboratory preparation of samples.

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