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Physiology of diapause in the adult bark beetle, Ips acuminatus Gyll., studied in relation to cold hardiness
J. lmecr Physiol. Vol. 31, No. 12, pp. 909-916, Printed in Great Britain. All rights reserved
1985 Copyright
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0022-1910/85 $3.00 + 0.00 1985 Pergamon Press Ltd
PHYSIOLOGY OF DIAPAUSE IN THE ADULT BARK BEETLE, IPS ACUMINATUS GYLL., STUDIED RELATION TO COLD HARDINESS
IN
UNN GEHRKEN Department of Biology, Division of Zoology, University of Oslo, P.O. Box 1050, Blindem, N-0316 Oslo 3, Norway (Received 22 January 1985: revised 24 April 1985)
Abstract-The ovarioles of the bark beetle, Ips acuminatus are telotrophic. Ovarian Pevelopment is suppressed at an immature stage with primary germ cells present in the germaria. Lower oxygen consumption is found in beetles during autumn and early winter, and a substantial rise in mean respiration
rates occurs in the beginning of January paralleled by a resumption of pre-vitellogenesis in all females maintained either at 3°C or out of doors after return to 21°C for 2 weeks. It is concluded that I. acuminatus enters faculative diapause soon after enclosure to the adult, and that diapause is terminated by mid-winter in beetles kept for 18 weeks at either 3°C or out of doors. The specimens remain thereafter in reproductive quiescence until ovarian development can proceed. Photic cues are neither involved in the elevation of mean respiration rates, nor needed to abolish the inhibition of ovarian maturation in beetles kept at 3°C or in those returned to 21°C. However, follicle formation in ovarioles is only seen in positive phototactic females reared during “long-day” conditions, suggesting a photoperiodic regulation of the later stages of vitellogenesis. Detectable amounts of ethylene glycol are found at the beginning of November in freezing-susceptible I. acuminatus hibernating in its galleries underneath bark of Scats pine (Pinus silvestris) at 3°C. The gradual catabolism of the cryoprotective solute at 3°C through December occurred at a time when individuals achieved the competence to resume ovarian maturation during 2 weeks at 21°C but prior to the substantial rise in their mean respiration rates. However, resumption of ovarian development in spring had no effect on the capability of outdoor beetles to enhance their supercooling capacity when subjected to sub-zero temperatures. Since the ability to respond to temperature changes occurred in post-diapause I. acuminatus as well, the maintenance of prolonged cold hardiness in specimens could not be related to diapause itself. Apparently, the ability of beetles to resynthesize ethylene glycol when a detectable level is present in the organism remains unaltered during overwintering. Key Word Index: Reproductive hardiness, Ips acuminatus
diapause,
pre-vitellogenesis,
INTRODUCTION
Overwintering in insects usually involves some form of dormancy. Dormancy is generally designated either quiescence or diapause (Lees, 1955). Simple quiescence is a suppression of developmental processes that is directly imposed by adverse physical conditions, and recovery occurs soon after conditions improve. Diapause is a genetically controlled suppression of development that typically begins before the onset of unfavourable conditions and may not be terminated until long after the disappearance of such conditions. Insects in diapause are usually identified on the basis of the negative criteria of suppressed developmental and metabolic rates. Respiratory metabolism is typically reduced during diapause and increased respiration rate is a reliable index of diapause termination (Lees, 1956; Siew, 1966; Tauber and Tauber, 1976). The pre-diapause reproductive system of newly emerged adult insects is usually in an early stage of development and by definition remains relatively undeveloped during the diapause period (Beck, 1980). However, much time may elapse between diapause termination and any detectable maturation of the germ cells (Tauber and Tauber, 1976).
ethylene glycol accumulation,
cold
A close correlation between diapause and accumulation of low molecular weight antifreeze has been reported in several insects (Chino, 1957; Somme, 1964, 1965; Asahina, 1966, Wyatt, 1969; Krunic and Salt, 1971; Mansingh and Smallman, 1972; Hansen, 1973; Mansingh, 1974). In several other diapausing species which undergo winter hardening, accumulation of cryoprotective compounds was not found to be attributable directly to diapause (Takehara, 1966; Baust and Miller, 1970; Ring, 1972; Zeiglet and Wyatt, 1975; Frankos and Platt, 1976; Wood and Nordin, 1976; Nordin et al., 1984). Accumulation of cryoprotective solutes at low temperatures occurs in non-diapausing insects as well, e.g. the carpenter ant, Camponotus pennsylvanicus (Dubach et al., 1959), Cumponotus herculeanus (Somme, 1964) and the bark beetle, Scolytus ratzeburgi (Ring, 1977). According to Salt (1961), cold hardiness and diapause are separate phenomena, the relation beteen the two arising from their concurrent timing. The bark beetle Zps acuminatus Gyll has only one generation a year in Scandinavia. In more southerly areas in Europe, however, the species is potentially multivoltine (Chararas, 1962). Only adults survive winter (Bakke, 1968), and low-temperature exposure does not promote accumulation of ethylene glycol in 909
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freezing susceptible adults until November (Gehrken, 1984). The aims of the research reported here were: (1) to determine whether adult I. acuminatus, during autumn and winter, possesses the physiological characteristics of diapause such as reduced respiration rate and suppression of ovarian development, (2) to elucidate whether the diapause condition was essential for maintenance of prolonged cold hardiness in beetles, and (3) to examine whether the ability of specimens to accumulate ethylene glycol upon low temperature exposure was changed during overwintering. MATERIALS
AND METHODS
Logs of Scats pine (Pinus silvestris) colonized by I. acuminatus were collected near Kongsberg (59”40’), SE Norway. After being brought to the University of Oslo in the end of August 1976 and again in 1982, logs were stored either out of doors under ambient conditions or at 3°C in the laboratory under continuous darkness. Supercooling point, concentration of ethylene glycol, respiration rate, water content, fresh weight and reproductive stage of beetles stored at 3°C as well as the fresh weight and reproductive stage of specimens maintained out of doors were examined regularly during the winter of 1976/77. The reproductive stage was determined immediately in one group whenever beetles were obtained from 3°C or out of doors, and after the following 2 weeks in a second group simultaneously returned to 21“C from both regimes. At 2 1“C, adults were housed in slightly moistened boxes with access to fresh bark chips, during continuous darkness. An experiment was conducted to determine lowtemperature influence on the rate of ethylene glycol accumulation in 1. acuminatus in spring. In April 1983, logs colonized with beetles were transferred to 21°C from the outdoor supply. Following 1 day of warm treatment, specimens were removed from logs and cold re-acclimated at -5’C for 5 days. The supercooling and haemolymph melting point, concentration of ethylene glycol, water content and reproductive stage were recorded immediately after beetles were obtained out of doors, after 1 day at 2 1“C and 5 days at - 5°C. The reproductive stage of beetles was, moreover, examined after 7 days at 2 1“C. 7 days at 21°C. Oxygen consumption rate Respiration rates were measured at 10, 15 and 20°C respectively with a constant-pressure respirometer (0.4 mm dia) described by Engelman (1963). Eleven replicate respirometers were used for each set of experiments. Three beetles were placed in each flask which had a filter paper saturated with 107; potassium hydroxide in the centre well to absorb carbon dioxide during respiration. Oxygen consumption was recorded as mm3 O,/mg fresh weight/h at STP and corrections were made for the partial pressure contributed by the 10% potassium hydroxide at the respective temperatures. Measured as a function of temperature, oxygen consumption was always determined from low to high temperatures in the
bath in each set of experiments. Beetles were allowed to equilibrate at each temperature for approx. 1 h prior to initiation or measurements. Readings were taken during consecutive intervals of 2&30min, depending on temperature in the bath and the rate of respiration. Empty respirometers served as thermo-barometric controls in all experiments. Dissection and histological technique
In the Kongsberg population of I. acuminatus, the sex ratio was about 1 male to 32 females, and only females were used. Reproductive stages were determined by dissection and by light microscopic studies of histological preparations of ovaries. Ovaries were dissected out in saline and graded according to the stages described by Hodek (1971). For light microscopic studies of histological preparations, ovaries were dissected out in Clarke’s solution (3: 1, absolute alcohol-acetic acid) and transferred to fresh fixative for 1 h at 20°C before being embedded in paraffin wax and sectioned (5pm). To distinguish between the pre-vitellogenetic stage 3 and the vitellogenetic stage 4, the histological procedure included the periodic acid-Schiff (PAS) technique both with and without 1% periodic oxidation as a general test for carbohydrates (Casselman, 1959). The nuclei were stained with Mayer’s acetic haematoxylin-eosine. The ovaries were considered to be in stage 4 when carbohydrate tests gave a PASpositive reaction due to deposition of carbohydrateprotein yolk. Differentiation of oocytes in the germaria (stage 2) were only seen occasionally in ovarioles of beetles during this investigation and were included with stage 1. For the purpose of the present paper, the results were given as percentage females of the whole sample with oocytes only present in germaria (stage 1 and 2). Sample sizes were generally between 20 and 30 females. Wuter content and fresh weight
The water content of beetles was determined following a procedure described by Gehrken (1984) and represented the mean of 6 individual measurements. Fresh weights were obtained from 3°C I. acuminatus after they had been used for respiration measurements and from outdoor specimens following supercooling-point determination. The mean fresh weight of beetles obtained from 3°C represented 30 individual measurements, and that of specimens sampled out of doors represented 12-18 individual measurements. Supercooling point, haemolymph melting point and solute concentration
The technique of measuring supercooling point, haemolymph melting point and ethylene glycol presence and quantity have been described by Gehrken (1984). Each determination of supercooling point of beetles obtained at 3°C represented the mean of 12 individual measurements and the concentration of ethylene glycol was the mean of 5 individual measurements. During cold re-acclimation of April beetles treated at 2 1“C for 1 day, each determination of supercooling points represented the mean of 7-21 individual measurements, haemolymph melting point was the mean
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Physiology of diapause in relation to cold hardiness
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Fig. 1.Oxygen consumption rates (mean f SEM) of fps ucuminatusmaintained at 3°C. monitored during experimental temperatures of IO, 15 and 20°C.
of 2-6, and ethylene glycol concentration mean of 3-9 measurements.
was the
Statistical methods The significance of differences between two means of supercooling points, haemolymph melting points, respiration rates, fresh weights, water contents and solute concentrations were assessed by Student’s ttest. Differences were considered significant when P 2 0.05.
RESULTS
Oxygen consumption rate The respiration rates measured in adult I. acuminatus stored at 3°C from the end of August 1976 until the end of April 1977 are presented in Fig. 1. Substantial higher mean oxygen consumptions at 10, 15 and 20°C were demonstrated from the beginning of January (P i 0.01) compared with those experienced 2 weeks earlier. A further rise in mean respiration rates was observed through January. From February onwards, the mean oxygen consumption of adults at the different experimental temperatures stabilized at the higher rates. Dissection and histological technique The ovarioles of I. acuminatus are telotrophic, and histological preparations revealed that “nutritive cords” connected various oiicytes with the apical trophic tissue. Each of the paired ovaries was composed of two semi-transparent ovarioles. They were structurally similar and their development seemed parallel. Outside the germaria, the number of oiicytes did not exceed two in each ovariole. Stage 5 and 6 of ovarian maturation were not observed in females unless given a chance to mate and establish galleries. Stage 1, in which the germaria contained primary germ cells, was the typical condition of ovaries
examined in September 1976. Differentiation into oijcytes in the vitellaria (stage 3) was not seen until the beginning of February in ovaries of beetles maintained at 3°C and a month later, at the beginning of March in ovaries of those stored out of doors (Fig. 2). In beetles transferred from the 3°C or the outdoor supplies in September and treated at 21°C the germaria became more opaque within 2 weeks. Differentiation into oijcytes found side by side among the pre-follicular cells at the anterior end of the vitellarium (stage 2) was only occasionally seen following warm treatment during October. In the end of November, differentiation into oijcytes in the vitellaria (stage 3) was seen in ovarioles of 7% of 3°C specimens returned to 21°C (Fig. 2). Through December, ovaries of a markedly increasing number of 3°C as well as outdoor beetles were graded stage 3 following warm treatment. From January onwards, all females had resumed ovarian development during 2 weeks at 21°C. Oiicytes containing yolk (stage 4) were not found in ovaries of beetles maintained at 3°C. However, deposition of yolk was demonstrated in ovarioles of about 70% of females kept out of doors and examined at the end of April. When transferred at the beginning of January from the 3°C or the outdoor supplies and kept at 21°C for 2 weeks, about one third of the females reached ovarian developmental stage 4. Only one oiicyte at a time seemed to be engaged in yolk deposition in each ovariole. In April 1983, obcytes were neither present in vitellaria of outdoor beetles, nor in those kept for 1 day at 21°C. Within 7 days of warm acclimation, however, beetles had emerged from logs and become positively phototactic. All positive phototactic females had initiated vitellogenesis, and when mated and given a chance to construct nuptial chambers, eggs were laid within a week at 21°C.
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Fig. 2. The effect of temperature on post-diapause maturation of ovaries of Ips acuminatus when obtained from 3’C or out of doors, and following 2 weeks at 2 1‘C during continuous darkness with access to fresh bark chips. The results are expressed as percentage females of the whole sample with only oiicytes present in the germaria (stage 1 and 2).
No extracellular spaces were visible in the follicular epithelium which surrounds the oacytes in ovaries of females returned to 21°C from the two regimes. Thus,
the developmental stage 4 in I. czcuminatus corresponds to an earlier stage of yolk deposition in the milkweed bug, Oncopeltus fasciatus (Schreiner, 1977) and the seed beetle, Bruchidius obectus (Biining, 1972). In ovarioles of positively phototactic I. ucuminatus, however, the follicular epithelium showed extracellular spaces. Dissection performed on beetles showed that the fat body was prominent in autumn and early winter, but diminished as the winter advanced. Water content and fresh
weight
A substantial loss in mean water content of beetles stored at 3°C occurred from the beginning of September until the beginning of November [P < 0.011 (Fig. 3). Through December, the mean water content of I. acuminatus was significantly elevated (P < 0.02) prior to the substantial increase in respiration rates. From January onwards, the body water of 3°C beetles remained stabilized. In females acclimated 1 day at 21°C in April 1983, the water content ranged from 56.6 to 49x, the mean however, was not significantly different compared to that of the initial sample obtained out of doors. The fresh weight of I. acuminatus during hibernation either at 3°C or out of doors is presented in Fig. 4. The mean body weight of specimens maintained at 3°C in the beginning of November was significantly lower than that of those examined in the end of August (P < 0.02). This loss paralleled the
reduction in mean water content of beetles (Fig. 3) and evacuation of food residues within the gut. A sudden change in mean fresh weight of 3°C specimens was seen in the beginning of January, when the body weight was significantly reduced compared to those weighed 2 weeks earlier (P < 0.01). From January onwards, no significant weight changes were observed, when the mean fresh weights monitored during this period were compared with the corresponding data obtained about 2 weeks later. The body weight of outdoor I. acuminatus subjected to environmental temperatures far below 0°C from December (Gehrken, 1984) decreased gradually during hibernation. By the beginning of March and resumption of ovarian development (Fig. 2), the weight loss was particularly pronounced (P < 0.001). The small weight increase demonctrated in beetles in the end of April, accompanied the resumption of feeding. Supercooling point, solute concentration
haemolymph
melting
point
and
The mean supercooling point of I. acuminatus maintained at 3” was depressed from - 17°C in the end of October to -23.8”C at the beginning of November (Fig. 3). A further insignificant depression of mean supercooling point was seen during November. The mean supercooling point was elevated through December and levelled off at about -20°C from January onwards. Ethylene glycol which, had been synthesized in beetles throughout November, was completely lost by early January (Fig. 3). Mannitol, sorbitol and dulcitol
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Physiology of diapause in relation to cold hardiness
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Fig. 3. Changes in water content, supercooling point and concentration of ethylene glycol (mean + SEM) of Ips acuminatus maintained at 3°C.
mean melting point by 3°C. When exposed to - 5°C for 5 days, a significant decrease in mean supercooling and haemolymph melting points (P < 0.01) occurred only in specimens capable of ethylene glycol accumulation.
produced in I. acuminates maintained out of doors (Gehrken, 1984) were not present in detectable amounts in specimens stored at 3°C. In April 1983, a complete depletion of ethylene glycol had taken place in one third of the beetles treated at 21°C for 1 day. An ability to re-accumulate the solute was only demonstrated in two thirds of specimens, which regained 75% of the initial amount during 5 days at - 5°C. In beetles treated 1 day at 21”C, the mean supercooling point was elevated by 5 to 7°C and the
DlSCUSSlON
The lower oxygen consumption rates of I. acuminatus (Fig. 1) and the failure of ovarian development in beetles stored at 3°C as well as in those
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Fig. 4. Changes in body weight (mean + SEM) of Ips acuminafus maintained at 3°C or out of doors
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returned to 21°C for 2 weeks (Fig. 2) could have resulted from either low-temperature exposure jn itself or from diapause. Beck (1980) pointed out that the onset of faculative diapause differs from that of quiescence in the capability of species to respond actively to periodic token stimuli that are not, in themselves, adverse. The fact that the suppression of ovarian development was seen even in outdoor females subjected to temperature favourable for growth, strongly suggests that I. acurninatus displays a facultative diapause during autumn and early winter. The higher respiration rates, from January onwards, indicated that diapause had been terminated in beetles kept at 3°C (Fig. 1). The elevation of respiratory rates corresponded with resumption of pre-vitellogenesis in 100% of females returned to 21°C for 2 weeks from either 3’C or out of doors (Fig. 2), suggests that reproductive diapause was completed in mid winter in outdoor beetles as well, before the onset of the coldest months. That hibernal diapause ends mid-winter rather than in spring is widely accepted (Tauber and Tauber, 1976; Beck, 1980). Ovarian maturation in I. acuminatus ceased at an early stage with primary germ cells present in germaria. Developmental arrest at an early stage of previtellogenesis has been reported in several diapausing beetles (Hodek and Cerkasov, 1961; Siew, 1966: Meidell, 1983). Apparently, 3°C was sufficient to allow previtellogenesis 1 month after diapause had been terminated (Fig. 2) but insufficient to promote yolk deposition. Tauber and Tauber (1973) also reported that pre-vitellogenesis can take place at such low temperatures and considered 4°C the approximate threshold for the earlier stages of oogenesis in the green lacewing, Crysopa carnea. The role of photoperiod in induction and termination of diapause was not studied in adult I. acuminatus. Adult diapause is commonly caused by exposure of the larval stages to a diapause-inducing photoperiod (Beck, 1980). De Wilde et al. (1959) reported that in diapausing Leptinotarsa decemlineata reared as larvae during long-day conditions, the ovaries contained ova in all stages of vitellogenesis. The Colorado potato beetle reared from the egg stage under short-day conditions, however, enter diapause with completely inactive vitellaria. Probably, I. acuminatus enter diapause shortly after emergence from the pupae due to diapause-inducing cues experienced at earlier stages of development. Photic cues were neither involved in elevation of oxygen consumption rates in beetles kept for 18 weeks at 3°C (Fig. I) nor needed to abolish inhibition of oiigenesis in females returned to 21°C followjng 12-16 weeks at 3°C (Fig. 2). Recently, Saunders (1983) reported that 12-14 weeks at 4°C overrode the critica daylength for diapause termination in the bug, Pyrrhocoris apterus and that eggs were laid at all temperatures when returned to “favourable” temperature. In Norway, 1. acuminatus endure 6 months of chilling temperatures, thus a photic cue for termination of diapause in the field would play no ecological role. The mature stages of vitellogenesis, however, seemed to be regulated by long-day conditions. DeWilde (1970) emphasized that an additional
factor released during long-day conditions was required to accomplish maintained vitellogenesis in the Colorado potato beetle. Ethylene glycol, doubtless, plays an important part in lowering of supercooling points of I. acuminatus after 2 months at 3°C (Fig. 3) [Gehrken, 19841. The thermal hysteresis factor proliferated during the same time (Gehrken, 1984) presumably lower the supercooling point by 3°C and explain stabilization of nucleating temperature at -20°C following a complete loss in ethylene glycol. A gradual catabolism of glycerol following diapause termination has been reported in several overwintering species (Sramme, 1982). The process of resynthesis of glycerol and sorbitol in eggs of Born& mori continued even when embryogenesis was suppressed at low temperature (Chino, 1957, 1960). In woolly bear larvae, Isis isabellu, high levels of glycerol were maintained during the activated phase of diapause when individuals had achieved the competence to resume development at suitable temperature (Mansingh and Smallman, 1972). In I. acuminatus, ethylene glycol was depleted at 3°C during the activated phase of reproductive diapause and had been completely catabolized in specimens increasing their respiratory rates. The restored physiological and biochemical process leading to resumption of ovarian development in beetles maintained out of doors by mid-April (Fig. 2) had no effect on their ability to enhance supercooling during 2 weeks at either -5’C or - 10°C (Gehrken, 1984). This strongly suggests that resynthesis of ethylene glycol at 3°C is neither specifically related to termination of diapause per se, nor with the process of post diapause pre-vitellogenesis in adult Z. acuminutus. The direct proof of the regulatory mechanisms to synthesis-resynthesis of ethylene glycol at 3°C is missing but might be found in temperaturedependent changes and/or respiratory processes. Once injtiated in November, accumulation of the diol is achieved at sub-zero temperatures (Gehrken, 1984), and synthesis is reversible only when detectable level is present in the organism. Complete depletion of ethylene glycol is probably accompanied by reversal of the extensive metabolic and enzymatic adjustments that are required for maintenance of high level of low molecular weight solute. Provided that ethylene glyco1 was present following warm acclimation 75-80x of the initial level was re-accumulated during 5 days at -5°C in December (Gehrken, 1984) as well as in April. The irreversible loss of the cryoprotective compound during 1 day at 21°C that occurred in one third of specimens in April, corresponded well to the consistently higher supercooling points of beetles in spring. Decrease in water content is probably an important part of becoming cold-hardy (Salt, 1961). I. acuminatus, either from out-of-doors (Gehrken, 1984) or kept at 3°C did not accumulate ethylene glycol until water content had dropped below 52%. The inability of specimens to re-synthesize the solute following a complete loss probably reflects a hydration-dependent trigger or control for ethylene glycol synthesis. Water content invariably increased following a complete loss in ethylene glycol in diapausing (Gehrken, 1984) as well as in post-
Physiology of diapause in relation to cold hardiness diapausing specimens. These findings agree with Kaufman (1971) who found that the outdoor Alaskan carbid beetle, Pterostichus brevicornis resumed feeding and ovarian development by mid-February, and the water content remained constant until the end of March, when the sharp rise paralleled a gradual depletion of glycerol (Baust and Miller, 1970). Frankos and Platt (1976) also found that the water content of Limenitis archippus larvae rose following depletion of glycerol though during diapause termination. Most investigators have concluded that a water increase alone does not seem likely to be a diapause-terminating factor (Beck, 1980). In experiments with the European corn borer larvae, Ostrinia nubilalis, it was found that the neuroendocrine system was not activated until the water requirement had been met (Beck, 1967). The increase in water content following a complete catabolism of ethylene glycol might be of vital importance to resume ovarian development in post-diapausing I. acuminatus. Whether initiation of ethylene glycol production arises from an enhanced temperature threshold for synthesis during diapause development or results from cues other than temperature such as photoperiod respiratory processes and/or water balance of individuals is not yet understood. However, low temperature is not the only cue providing the definitive signal to initiate accumulation of the cryoprotective diol in beetles, and the triggers to winter hardening of I. acuminatus will be of more than one class. That the body weight of insects stabilized in the cold following the first weeks, have been emphasized (Kaufman, 1971; Wood and Nordin, 1976). In I. acuminatus, the substantial weight loss that occurred after 18 weeks at 3°C corresponded well to increased metabolic rates in specimens upon diapause termination. Between the termination of diapause and mid-April, the cold hardy outdoor beetles had lost twice as much weight as the 3°C insects lacking ethylene glycol in their body fluid. During this period, the outdoor specimens had not experienced high enough temperatures to resume feeding or to initiate vitellogenesis. The difference in weight loss between groups might therefore, at least in part, be due to the outdoor beetles having to manufacture presumed metabolically costly cryoprotective solutes. This study reveals that the potentially multivoltine I. acuminatus displays a facultative diapause during autumn and early winter, characterized by reduced respiration rates, cessation of ovarian maturation at an immature pre-vitellogenic stage, decline in water content and prominent fat body. Detectable amounts of ethylene glycol in 3°C beetles occurred during the same time as in the outdoor insects during diapause. Provided that the diol was not completely catabolized, the rate of accumulation at sub-zero temperatures seemed unaltered during hibernation. Evidently, diapause condition is not essential for the maintenance of prolonged cold hardiness, and beetles respond to temperature changes below 0°C during post-diapause maturation of ovaries. An early termination of diapause combined with an apparently low temperature threshold for pre-vitellogenesis allow adult I. acuminatus to take full advantage of
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the occasional warm periods in early spring. This is an important ability when the growing season is predictably very short. Acknowledgements-I am most grateful to Dr L. Ssmme for valuable advice throughout the present investigation, and to Mrs I. Tambs-Lyche for doing the paraffin embedding and the sectioning. I thank Mrs B. Schreiner for valuable advice in evaluation of the histological preparations.
REFERENCES
Asahina E. (1966) Freezing and frost resistance in insects. In Cryobiology (Ed. by Meryman H. T.), pp. 451-486. Academic Press, New York. Bakke A. (1968) Ecological studies on bark beetles (Coleoptera: Scolytidae) associated with Scats pine (Pinus sihestris L.) in Norway with particular reference to the influence of temperature. Meddr. norske Skogfbrs. Ves. 21, 441-602. Baust J. G. and Miller L. K. (1970) Variation in glycerol content and its influence on cold hardiness in Alaskan carabid beetle, Pterostichus brevicornis. 1. Insect Phwiol. 16, 979-990. Beck S. D. (1967) Water intake and the termination of diapause in the European corn borer. Ostrinia nubilulis. J. Insect Physiol. 13, 739-750. Beck S. D. (1980) Znsecr Photoperiodism. 2nd edn. Academic Press, London. Biining J. (1972) Untersuchung am Ovar von Bruchidius obectus Say. (Coleoptera-Polyphaga) zur Kkirung des Oocytenwachstums in der PrCviteliogene. Z. Zellf&ch. 128, 241-282. Casselman W. G. (1959) Histochemical Technique. Methuen, London. Chararas C. (1962) Etude Bioloqique des Scolytide,y des Coniferes (Ed. by Lechevalier P.). Paris. Chino H. (1957) Conversion of glycogen to sorbitol and glycerol in the diapause egg of the Bon&x silkworm. Nature 180, 60&607. Chino H. (1960) Enzymatic pathways in the formation of sorbitol and glycerol in the diapausing eggs of the silkworm Bombyx mori. 1: On the polyol dehydrogenases. J. Insect Physiol. 5, I-15. DeWilde J. (1970) Hormonal control of synthesis of vitellogenic female protein in the Colorado beetle, Leptinotarsa decemlineata. J. Insect Physiol. 16, 1455-1466. DeWilde J., Duintjer C. S. and Mook L. (1959)Physiology and diapause in the adult Colorado beetle (Leptinotarsa decemlineata SaybI. The photoperiod as a controlling factor. J. insect Physiol. 3, 75-85. Dubach P., Pratt D.. Smith F. and Stewart C. M. (1959) Possible role of glycerol on the winter-hardiness of insects. Nature 184, 288-289. Engelman M. D. (1963) A constant pressure respirometer for small arthropods. Enf. News 74, 181-186. Frankos V. H. and Platt A. P. (1976) Glycerol accumulation and water content in larvae of Limenitis archippus: their importance to winter survival. J. Insect Physiol. 22, 632-638. Gehrken U. (1984) Winter survival of an adult bark beetle, Ips acuminatus Gyll. J. Insect Physiof. 30, 421-429. Hansen T. (1973) Variation in glycerol content in relation to cold-hardiness in the larvae of Petrova resinella L. (Lepidoptera, Tortricidae). Eesti NSV Tead. Akad. Toim. 22, 105-112. Hodek I. (1971) Induction of adult diapause in Pyrrhocoris apterus L. by short cold exposure. Cecofogia 6, 109-l 17. Hodek I. and Cerkasov J. (1961) Prevention and artificial induction of imaginal diapause in Coccinella septempunctata L. (Col., Coccinellidae). Entomologia exp. appl. 4, 179-190.
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Kaufman T. (1971) Hibernation in the arctic beetle, Pterostichus brevicornis, in Alaska. J. Kans ent. Sot. 44, 8 l-92. Krunic M. D. and Salt R. W. (1971) Seasonal changes in glycerol content and supercooling points of Megachile rotundata (F.) and M. relativa Cress. Can. J. 2001. 49, 663-666. Lees A. D. (1955) The Physiology ofDiapause in Arthropods. Cambridge University Press, New York. Lees A. D. (1956) The physiology and biochemistry of diapause. A. Rec. Ent. 1, I-16. Mansingh A. (1974) Studies on insect dormancy. II. Relation of cold-hardiness to diapause and quiescence in the eastern tent caterpillar, Malacosoma americanum (Fabr.), (Lasiocamphidae: Lepidoptera). Can. J. 2001.52, 629-637. Mansingh A. and Smallman B. N. (1972) Variation in polyhydric alcohol in relation to diapause and coldhardiness in the larvae of Isis isabella. J. Insect Phvsiol. 18, 1565-1.571. Meidell E.-M. (1983) Diapause, aerobic and anaerobic metabolism in alpine, adult Melasoma collaris (Coleoptera). Oikos 41, 239-244. Nordin J. H., Zheng C. and Chih-Ming Y. (1984) Coldinduced glycerol accumulation by Ostrinia nubilalis larvae is developmentally regulated. 30, 563-566. Ring R. A. (1972) Relationship between diapause and supercooling in the blowfly Lucilia sericata (Mg.) (Diptera: Calliphoridae). Can. J. Zool. 50, 1601-1605. Ring R. A. (1977) Cold-hardiness of the bark beetle. SFolytus rat_ebu& Jans. (Cal.. Scolytidae). Norm. J. Ent. 24, 125-136. Salt R. W. (1961) Principles of insect cold-hardiness. A. Rer. Ent. 6, 55-74. Salt R. W. (1969) The survival of insects at low temperatures. In Dormancy and Suwiral. Svmp. Sot. exp. Biol. 23, 331-350.
c
Saunders D. S. (1983) Diapause induction-termination asymmetry in the photoperiodic responses of the linden bug, Pyrrhocoris apterus and an effect of near-critical photoperiods on development. J. Insect Ph_vsiol. 29, 399405. Schreiner B. (1977) Vitellogenesis in the milkweed bug, Oncopelfus fasciatus Dallas (Hemiptera). A light and electron microscopic investigation. J. Morph. 151, 35-80. Siew Y. C. (1966) Some physiological aspects of adult reproductive diapause in Galeruca tanaceti (L.) (Coleoptera: Crysomelidae). Trans. R. ent. Sot. Lond. 118, 359-374. Snmme L. (1964) Effects of glycerol on cold-hardiness in insects. Can. J. Zoo/. 42, 89-101. Ssmme L. (1965) Further observations on glycerol and cold-hardiness in insects. Can. J. 2001. 43, 765-770. Ssmme L. (1982) Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. 73A, 519-543. Takehara I. (1966)Natural occurrence of glycerol in the slug caterpillar. Monemajavescens. Low Temp. Sci. Ser. B 14, l-34. Tauber M. J. and Tauber C. A. (1973) Seasonal regulation of dormancy in Chrysopa carnea (Neuroptera). J. Insect Physiol. 19, 1455-14631 Tauber M. J. and Tauber C. A. (1976) Insect seasonality: diapause maintenance, termination. and postdiapause development. A. Ren. En?. 21, 81-107. Wood F. E. and Nordin J. H. (1976) Studies on low temperature induced biogenesis oi glyckrol by adult Protophormia terranocae. J. Insect Physiol. 22, 1665-1673. Wyatt G. R. (1969) The biochemistry of sugars and polysaccharides in insects. Adv. Insect Physiol. 4, 287-360. Ziegler R. and Wyatt G. R. (1975) Phosphorylase and glycerol production activated by cold in diapausing silkmoth pupae. Nature 254, 622-623.
1985 Copyright
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0022-1910/85 $3.00 + 0.00 1985 Pergamon Press Ltd
PHYSIOLOGY OF DIAPAUSE IN THE ADULT BARK BEETLE, IPS ACUMINATUS GYLL., STUDIED RELATION TO COLD HARDINESS
IN
UNN GEHRKEN Department of Biology, Division of Zoology, University of Oslo, P.O. Box 1050, Blindem, N-0316 Oslo 3, Norway (Received 22 January 1985: revised 24 April 1985)
Abstract-The ovarioles of the bark beetle, Ips acuminatus are telotrophic. Ovarian Pevelopment is suppressed at an immature stage with primary germ cells present in the germaria. Lower oxygen consumption is found in beetles during autumn and early winter, and a substantial rise in mean respiration
rates occurs in the beginning of January paralleled by a resumption of pre-vitellogenesis in all females maintained either at 3°C or out of doors after return to 21°C for 2 weeks. It is concluded that I. acuminatus enters faculative diapause soon after enclosure to the adult, and that diapause is terminated by mid-winter in beetles kept for 18 weeks at either 3°C or out of doors. The specimens remain thereafter in reproductive quiescence until ovarian development can proceed. Photic cues are neither involved in the elevation of mean respiration rates, nor needed to abolish the inhibition of ovarian maturation in beetles kept at 3°C or in those returned to 21°C. However, follicle formation in ovarioles is only seen in positive phototactic females reared during “long-day” conditions, suggesting a photoperiodic regulation of the later stages of vitellogenesis. Detectable amounts of ethylene glycol are found at the beginning of November in freezing-susceptible I. acuminatus hibernating in its galleries underneath bark of Scats pine (Pinus silvestris) at 3°C. The gradual catabolism of the cryoprotective solute at 3°C through December occurred at a time when individuals achieved the competence to resume ovarian maturation during 2 weeks at 21°C but prior to the substantial rise in their mean respiration rates. However, resumption of ovarian development in spring had no effect on the capability of outdoor beetles to enhance their supercooling capacity when subjected to sub-zero temperatures. Since the ability to respond to temperature changes occurred in post-diapause I. acuminatus as well, the maintenance of prolonged cold hardiness in specimens could not be related to diapause itself. Apparently, the ability of beetles to resynthesize ethylene glycol when a detectable level is present in the organism remains unaltered during overwintering. Key Word Index: Reproductive hardiness, Ips acuminatus
diapause,
pre-vitellogenesis,
INTRODUCTION
Overwintering in insects usually involves some form of dormancy. Dormancy is generally designated either quiescence or diapause (Lees, 1955). Simple quiescence is a suppression of developmental processes that is directly imposed by adverse physical conditions, and recovery occurs soon after conditions improve. Diapause is a genetically controlled suppression of development that typically begins before the onset of unfavourable conditions and may not be terminated until long after the disappearance of such conditions. Insects in diapause are usually identified on the basis of the negative criteria of suppressed developmental and metabolic rates. Respiratory metabolism is typically reduced during diapause and increased respiration rate is a reliable index of diapause termination (Lees, 1956; Siew, 1966; Tauber and Tauber, 1976). The pre-diapause reproductive system of newly emerged adult insects is usually in an early stage of development and by definition remains relatively undeveloped during the diapause period (Beck, 1980). However, much time may elapse between diapause termination and any detectable maturation of the germ cells (Tauber and Tauber, 1976).
ethylene glycol accumulation,
cold
A close correlation between diapause and accumulation of low molecular weight antifreeze has been reported in several insects (Chino, 1957; Somme, 1964, 1965; Asahina, 1966, Wyatt, 1969; Krunic and Salt, 1971; Mansingh and Smallman, 1972; Hansen, 1973; Mansingh, 1974). In several other diapausing species which undergo winter hardening, accumulation of cryoprotective compounds was not found to be attributable directly to diapause (Takehara, 1966; Baust and Miller, 1970; Ring, 1972; Zeiglet and Wyatt, 1975; Frankos and Platt, 1976; Wood and Nordin, 1976; Nordin et al., 1984). Accumulation of cryoprotective solutes at low temperatures occurs in non-diapausing insects as well, e.g. the carpenter ant, Camponotus pennsylvanicus (Dubach et al., 1959), Cumponotus herculeanus (Somme, 1964) and the bark beetle, Scolytus ratzeburgi (Ring, 1977). According to Salt (1961), cold hardiness and diapause are separate phenomena, the relation beteen the two arising from their concurrent timing. The bark beetle Zps acuminatus Gyll has only one generation a year in Scandinavia. In more southerly areas in Europe, however, the species is potentially multivoltine (Chararas, 1962). Only adults survive winter (Bakke, 1968), and low-temperature exposure does not promote accumulation of ethylene glycol in 909
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freezing susceptible adults until November (Gehrken, 1984). The aims of the research reported here were: (1) to determine whether adult I. acuminatus, during autumn and winter, possesses the physiological characteristics of diapause such as reduced respiration rate and suppression of ovarian development, (2) to elucidate whether the diapause condition was essential for maintenance of prolonged cold hardiness in beetles, and (3) to examine whether the ability of specimens to accumulate ethylene glycol upon low temperature exposure was changed during overwintering. MATERIALS
AND METHODS
Logs of Scats pine (Pinus silvestris) colonized by I. acuminatus were collected near Kongsberg (59”40’), SE Norway. After being brought to the University of Oslo in the end of August 1976 and again in 1982, logs were stored either out of doors under ambient conditions or at 3°C in the laboratory under continuous darkness. Supercooling point, concentration of ethylene glycol, respiration rate, water content, fresh weight and reproductive stage of beetles stored at 3°C as well as the fresh weight and reproductive stage of specimens maintained out of doors were examined regularly during the winter of 1976/77. The reproductive stage was determined immediately in one group whenever beetles were obtained from 3°C or out of doors, and after the following 2 weeks in a second group simultaneously returned to 21“C from both regimes. At 2 1“C, adults were housed in slightly moistened boxes with access to fresh bark chips, during continuous darkness. An experiment was conducted to determine lowtemperature influence on the rate of ethylene glycol accumulation in 1. acuminatus in spring. In April 1983, logs colonized with beetles were transferred to 21°C from the outdoor supply. Following 1 day of warm treatment, specimens were removed from logs and cold re-acclimated at -5’C for 5 days. The supercooling and haemolymph melting point, concentration of ethylene glycol, water content and reproductive stage were recorded immediately after beetles were obtained out of doors, after 1 day at 2 1“C and 5 days at - 5°C. The reproductive stage of beetles was, moreover, examined after 7 days at 2 1“C. 7 days at 21°C. Oxygen consumption rate Respiration rates were measured at 10, 15 and 20°C respectively with a constant-pressure respirometer (0.4 mm dia) described by Engelman (1963). Eleven replicate respirometers were used for each set of experiments. Three beetles were placed in each flask which had a filter paper saturated with 107; potassium hydroxide in the centre well to absorb carbon dioxide during respiration. Oxygen consumption was recorded as mm3 O,/mg fresh weight/h at STP and corrections were made for the partial pressure contributed by the 10% potassium hydroxide at the respective temperatures. Measured as a function of temperature, oxygen consumption was always determined from low to high temperatures in the
bath in each set of experiments. Beetles were allowed to equilibrate at each temperature for approx. 1 h prior to initiation or measurements. Readings were taken during consecutive intervals of 2&30min, depending on temperature in the bath and the rate of respiration. Empty respirometers served as thermo-barometric controls in all experiments. Dissection and histological technique
In the Kongsberg population of I. acuminatus, the sex ratio was about 1 male to 32 females, and only females were used. Reproductive stages were determined by dissection and by light microscopic studies of histological preparations of ovaries. Ovaries were dissected out in saline and graded according to the stages described by Hodek (1971). For light microscopic studies of histological preparations, ovaries were dissected out in Clarke’s solution (3: 1, absolute alcohol-acetic acid) and transferred to fresh fixative for 1 h at 20°C before being embedded in paraffin wax and sectioned (5pm). To distinguish between the pre-vitellogenetic stage 3 and the vitellogenetic stage 4, the histological procedure included the periodic acid-Schiff (PAS) technique both with and without 1% periodic oxidation as a general test for carbohydrates (Casselman, 1959). The nuclei were stained with Mayer’s acetic haematoxylin-eosine. The ovaries were considered to be in stage 4 when carbohydrate tests gave a PASpositive reaction due to deposition of carbohydrateprotein yolk. Differentiation of oocytes in the germaria (stage 2) were only seen occasionally in ovarioles of beetles during this investigation and were included with stage 1. For the purpose of the present paper, the results were given as percentage females of the whole sample with oocytes only present in germaria (stage 1 and 2). Sample sizes were generally between 20 and 30 females. Wuter content and fresh weight
The water content of beetles was determined following a procedure described by Gehrken (1984) and represented the mean of 6 individual measurements. Fresh weights were obtained from 3°C I. acuminatus after they had been used for respiration measurements and from outdoor specimens following supercooling-point determination. The mean fresh weight of beetles obtained from 3°C represented 30 individual measurements, and that of specimens sampled out of doors represented 12-18 individual measurements. Supercooling point, haemolymph melting point and solute concentration
The technique of measuring supercooling point, haemolymph melting point and ethylene glycol presence and quantity have been described by Gehrken (1984). Each determination of supercooling point of beetles obtained at 3°C represented the mean of 12 individual measurements and the concentration of ethylene glycol was the mean of 5 individual measurements. During cold re-acclimation of April beetles treated at 2 1“C for 1 day, each determination of supercooling points represented the mean of 7-21 individual measurements, haemolymph melting point was the mean
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Physiology of diapause in relation to cold hardiness
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Fig. 1.Oxygen consumption rates (mean f SEM) of fps ucuminatusmaintained at 3°C. monitored during experimental temperatures of IO, 15 and 20°C.
of 2-6, and ethylene glycol concentration mean of 3-9 measurements.
was the
Statistical methods The significance of differences between two means of supercooling points, haemolymph melting points, respiration rates, fresh weights, water contents and solute concentrations were assessed by Student’s ttest. Differences were considered significant when P 2 0.05.
RESULTS
Oxygen consumption rate The respiration rates measured in adult I. acuminatus stored at 3°C from the end of August 1976 until the end of April 1977 are presented in Fig. 1. Substantial higher mean oxygen consumptions at 10, 15 and 20°C were demonstrated from the beginning of January (P i 0.01) compared with those experienced 2 weeks earlier. A further rise in mean respiration rates was observed through January. From February onwards, the mean oxygen consumption of adults at the different experimental temperatures stabilized at the higher rates. Dissection and histological technique The ovarioles of I. acuminatus are telotrophic, and histological preparations revealed that “nutritive cords” connected various oiicytes with the apical trophic tissue. Each of the paired ovaries was composed of two semi-transparent ovarioles. They were structurally similar and their development seemed parallel. Outside the germaria, the number of oiicytes did not exceed two in each ovariole. Stage 5 and 6 of ovarian maturation were not observed in females unless given a chance to mate and establish galleries. Stage 1, in which the germaria contained primary germ cells, was the typical condition of ovaries
examined in September 1976. Differentiation into oijcytes in the vitellaria (stage 3) was not seen until the beginning of February in ovaries of beetles maintained at 3°C and a month later, at the beginning of March in ovaries of those stored out of doors (Fig. 2). In beetles transferred from the 3°C or the outdoor supplies in September and treated at 21°C the germaria became more opaque within 2 weeks. Differentiation into oijcytes found side by side among the pre-follicular cells at the anterior end of the vitellarium (stage 2) was only occasionally seen following warm treatment during October. In the end of November, differentiation into oijcytes in the vitellaria (stage 3) was seen in ovarioles of 7% of 3°C specimens returned to 21°C (Fig. 2). Through December, ovaries of a markedly increasing number of 3°C as well as outdoor beetles were graded stage 3 following warm treatment. From January onwards, all females had resumed ovarian development during 2 weeks at 21°C. Oiicytes containing yolk (stage 4) were not found in ovaries of beetles maintained at 3°C. However, deposition of yolk was demonstrated in ovarioles of about 70% of females kept out of doors and examined at the end of April. When transferred at the beginning of January from the 3°C or the outdoor supplies and kept at 21°C for 2 weeks, about one third of the females reached ovarian developmental stage 4. Only one oiicyte at a time seemed to be engaged in yolk deposition in each ovariole. In April 1983, obcytes were neither present in vitellaria of outdoor beetles, nor in those kept for 1 day at 21°C. Within 7 days of warm acclimation, however, beetles had emerged from logs and become positively phototactic. All positive phototactic females had initiated vitellogenesis, and when mated and given a chance to construct nuptial chambers, eggs were laid within a week at 21°C.
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Month
Fig. 2. The effect of temperature on post-diapause maturation of ovaries of Ips acuminatus when obtained from 3’C or out of doors, and following 2 weeks at 2 1‘C during continuous darkness with access to fresh bark chips. The results are expressed as percentage females of the whole sample with only oiicytes present in the germaria (stage 1 and 2).
No extracellular spaces were visible in the follicular epithelium which surrounds the oacytes in ovaries of females returned to 21°C from the two regimes. Thus,
the developmental stage 4 in I. czcuminatus corresponds to an earlier stage of yolk deposition in the milkweed bug, Oncopeltus fasciatus (Schreiner, 1977) and the seed beetle, Bruchidius obectus (Biining, 1972). In ovarioles of positively phototactic I. ucuminatus, however, the follicular epithelium showed extracellular spaces. Dissection performed on beetles showed that the fat body was prominent in autumn and early winter, but diminished as the winter advanced. Water content and fresh
weight
A substantial loss in mean water content of beetles stored at 3°C occurred from the beginning of September until the beginning of November [P < 0.011 (Fig. 3). Through December, the mean water content of I. acuminatus was significantly elevated (P < 0.02) prior to the substantial increase in respiration rates. From January onwards, the body water of 3°C beetles remained stabilized. In females acclimated 1 day at 21°C in April 1983, the water content ranged from 56.6 to 49x, the mean however, was not significantly different compared to that of the initial sample obtained out of doors. The fresh weight of I. acuminatus during hibernation either at 3°C or out of doors is presented in Fig. 4. The mean body weight of specimens maintained at 3°C in the beginning of November was significantly lower than that of those examined in the end of August (P < 0.02). This loss paralleled the
reduction in mean water content of beetles (Fig. 3) and evacuation of food residues within the gut. A sudden change in mean fresh weight of 3°C specimens was seen in the beginning of January, when the body weight was significantly reduced compared to those weighed 2 weeks earlier (P < 0.01). From January onwards, no significant weight changes were observed, when the mean fresh weights monitored during this period were compared with the corresponding data obtained about 2 weeks later. The body weight of outdoor I. acuminatus subjected to environmental temperatures far below 0°C from December (Gehrken, 1984) decreased gradually during hibernation. By the beginning of March and resumption of ovarian development (Fig. 2), the weight loss was particularly pronounced (P < 0.001). The small weight increase demonctrated in beetles in the end of April, accompanied the resumption of feeding. Supercooling point, solute concentration
haemolymph
melting
point
and
The mean supercooling point of I. acuminatus maintained at 3” was depressed from - 17°C in the end of October to -23.8”C at the beginning of November (Fig. 3). A further insignificant depression of mean supercooling point was seen during November. The mean supercooling point was elevated through December and levelled off at about -20°C from January onwards. Ethylene glycol which, had been synthesized in beetles throughout November, was completely lost by early January (Fig. 3). Mannitol, sorbitol and dulcitol
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Physiology of diapause in relation to cold hardiness
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Fig. 3. Changes in water content, supercooling point and concentration of ethylene glycol (mean + SEM) of Ips acuminatus maintained at 3°C.
mean melting point by 3°C. When exposed to - 5°C for 5 days, a significant decrease in mean supercooling and haemolymph melting points (P < 0.01) occurred only in specimens capable of ethylene glycol accumulation.
produced in I. acuminates maintained out of doors (Gehrken, 1984) were not present in detectable amounts in specimens stored at 3°C. In April 1983, a complete depletion of ethylene glycol had taken place in one third of the beetles treated at 21°C for 1 day. An ability to re-accumulate the solute was only demonstrated in two thirds of specimens, which regained 75% of the initial amount during 5 days at - 5°C. In beetles treated 1 day at 21”C, the mean supercooling point was elevated by 5 to 7°C and the
DlSCUSSlON
The lower oxygen consumption rates of I. acuminatus (Fig. 1) and the failure of ovarian development in beetles stored at 3°C as well as in those
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Fig. 4. Changes in body weight (mean + SEM) of Ips acuminafus maintained at 3°C or out of doors
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returned to 21°C for 2 weeks (Fig. 2) could have resulted from either low-temperature exposure jn itself or from diapause. Beck (1980) pointed out that the onset of faculative diapause differs from that of quiescence in the capability of species to respond actively to periodic token stimuli that are not, in themselves, adverse. The fact that the suppression of ovarian development was seen even in outdoor females subjected to temperature favourable for growth, strongly suggests that I. acurninatus displays a facultative diapause during autumn and early winter. The higher respiration rates, from January onwards, indicated that diapause had been terminated in beetles kept at 3°C (Fig. 1). The elevation of respiratory rates corresponded with resumption of pre-vitellogenesis in 100% of females returned to 21°C for 2 weeks from either 3’C or out of doors (Fig. 2), suggests that reproductive diapause was completed in mid winter in outdoor beetles as well, before the onset of the coldest months. That hibernal diapause ends mid-winter rather than in spring is widely accepted (Tauber and Tauber, 1976; Beck, 1980). Ovarian maturation in I. acuminatus ceased at an early stage with primary germ cells present in germaria. Developmental arrest at an early stage of previtellogenesis has been reported in several diapausing beetles (Hodek and Cerkasov, 1961; Siew, 1966: Meidell, 1983). Apparently, 3°C was sufficient to allow previtellogenesis 1 month after diapause had been terminated (Fig. 2) but insufficient to promote yolk deposition. Tauber and Tauber (1973) also reported that pre-vitellogenesis can take place at such low temperatures and considered 4°C the approximate threshold for the earlier stages of oogenesis in the green lacewing, Crysopa carnea. The role of photoperiod in induction and termination of diapause was not studied in adult I. acuminatus. Adult diapause is commonly caused by exposure of the larval stages to a diapause-inducing photoperiod (Beck, 1980). De Wilde et al. (1959) reported that in diapausing Leptinotarsa decemlineata reared as larvae during long-day conditions, the ovaries contained ova in all stages of vitellogenesis. The Colorado potato beetle reared from the egg stage under short-day conditions, however, enter diapause with completely inactive vitellaria. Probably, I. acuminatus enter diapause shortly after emergence from the pupae due to diapause-inducing cues experienced at earlier stages of development. Photic cues were neither involved in elevation of oxygen consumption rates in beetles kept for 18 weeks at 3°C (Fig. I) nor needed to abolish inhibition of oiigenesis in females returned to 21°C followjng 12-16 weeks at 3°C (Fig. 2). Recently, Saunders (1983) reported that 12-14 weeks at 4°C overrode the critica daylength for diapause termination in the bug, Pyrrhocoris apterus and that eggs were laid at all temperatures when returned to “favourable” temperature. In Norway, 1. acuminatus endure 6 months of chilling temperatures, thus a photic cue for termination of diapause in the field would play no ecological role. The mature stages of vitellogenesis, however, seemed to be regulated by long-day conditions. DeWilde (1970) emphasized that an additional
factor released during long-day conditions was required to accomplish maintained vitellogenesis in the Colorado potato beetle. Ethylene glycol, doubtless, plays an important part in lowering of supercooling points of I. acuminatus after 2 months at 3°C (Fig. 3) [Gehrken, 19841. The thermal hysteresis factor proliferated during the same time (Gehrken, 1984) presumably lower the supercooling point by 3°C and explain stabilization of nucleating temperature at -20°C following a complete loss in ethylene glycol. A gradual catabolism of glycerol following diapause termination has been reported in several overwintering species (Sramme, 1982). The process of resynthesis of glycerol and sorbitol in eggs of Born& mori continued even when embryogenesis was suppressed at low temperature (Chino, 1957, 1960). In woolly bear larvae, Isis isabellu, high levels of glycerol were maintained during the activated phase of diapause when individuals had achieved the competence to resume development at suitable temperature (Mansingh and Smallman, 1972). In I. acuminatus, ethylene glycol was depleted at 3°C during the activated phase of reproductive diapause and had been completely catabolized in specimens increasing their respiratory rates. The restored physiological and biochemical process leading to resumption of ovarian development in beetles maintained out of doors by mid-April (Fig. 2) had no effect on their ability to enhance supercooling during 2 weeks at either -5’C or - 10°C (Gehrken, 1984). This strongly suggests that resynthesis of ethylene glycol at 3°C is neither specifically related to termination of diapause per se, nor with the process of post diapause pre-vitellogenesis in adult Z. acuminutus. The direct proof of the regulatory mechanisms to synthesis-resynthesis of ethylene glycol at 3°C is missing but might be found in temperaturedependent changes and/or respiratory processes. Once injtiated in November, accumulation of the diol is achieved at sub-zero temperatures (Gehrken, 1984), and synthesis is reversible only when detectable level is present in the organism. Complete depletion of ethylene glycol is probably accompanied by reversal of the extensive metabolic and enzymatic adjustments that are required for maintenance of high level of low molecular weight solute. Provided that ethylene glyco1 was present following warm acclimation 75-80x of the initial level was re-accumulated during 5 days at -5°C in December (Gehrken, 1984) as well as in April. The irreversible loss of the cryoprotective compound during 1 day at 21°C that occurred in one third of specimens in April, corresponded well to the consistently higher supercooling points of beetles in spring. Decrease in water content is probably an important part of becoming cold-hardy (Salt, 1961). I. acuminatus, either from out-of-doors (Gehrken, 1984) or kept at 3°C did not accumulate ethylene glycol until water content had dropped below 52%. The inability of specimens to re-synthesize the solute following a complete loss probably reflects a hydration-dependent trigger or control for ethylene glycol synthesis. Water content invariably increased following a complete loss in ethylene glycol in diapausing (Gehrken, 1984) as well as in post-
Physiology of diapause in relation to cold hardiness diapausing specimens. These findings agree with Kaufman (1971) who found that the outdoor Alaskan carbid beetle, Pterostichus brevicornis resumed feeding and ovarian development by mid-February, and the water content remained constant until the end of March, when the sharp rise paralleled a gradual depletion of glycerol (Baust and Miller, 1970). Frankos and Platt (1976) also found that the water content of Limenitis archippus larvae rose following depletion of glycerol though during diapause termination. Most investigators have concluded that a water increase alone does not seem likely to be a diapause-terminating factor (Beck, 1980). In experiments with the European corn borer larvae, Ostrinia nubilalis, it was found that the neuroendocrine system was not activated until the water requirement had been met (Beck, 1967). The increase in water content following a complete catabolism of ethylene glycol might be of vital importance to resume ovarian development in post-diapausing I. acuminatus. Whether initiation of ethylene glycol production arises from an enhanced temperature threshold for synthesis during diapause development or results from cues other than temperature such as photoperiod respiratory processes and/or water balance of individuals is not yet understood. However, low temperature is not the only cue providing the definitive signal to initiate accumulation of the cryoprotective diol in beetles, and the triggers to winter hardening of I. acuminatus will be of more than one class. That the body weight of insects stabilized in the cold following the first weeks, have been emphasized (Kaufman, 1971; Wood and Nordin, 1976). In I. acuminatus, the substantial weight loss that occurred after 18 weeks at 3°C corresponded well to increased metabolic rates in specimens upon diapause termination. Between the termination of diapause and mid-April, the cold hardy outdoor beetles had lost twice as much weight as the 3°C insects lacking ethylene glycol in their body fluid. During this period, the outdoor specimens had not experienced high enough temperatures to resume feeding or to initiate vitellogenesis. The difference in weight loss between groups might therefore, at least in part, be due to the outdoor beetles having to manufacture presumed metabolically costly cryoprotective solutes. This study reveals that the potentially multivoltine I. acuminatus displays a facultative diapause during autumn and early winter, characterized by reduced respiration rates, cessation of ovarian maturation at an immature pre-vitellogenic stage, decline in water content and prominent fat body. Detectable amounts of ethylene glycol in 3°C beetles occurred during the same time as in the outdoor insects during diapause. Provided that the diol was not completely catabolized, the rate of accumulation at sub-zero temperatures seemed unaltered during hibernation. Evidently, diapause condition is not essential for the maintenance of prolonged cold hardiness, and beetles respond to temperature changes below 0°C during post-diapause maturation of ovaries. An early termination of diapause combined with an apparently low temperature threshold for pre-vitellogenesis allow adult I. acuminatus to take full advantage of
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the occasional warm periods in early spring. This is an important ability when the growing season is predictably very short. Acknowledgements-I am most grateful to Dr L. Ssmme for valuable advice throughout the present investigation, and to Mrs I. Tambs-Lyche for doing the paraffin embedding and the sectioning. I thank Mrs B. Schreiner for valuable advice in evaluation of the histological preparations.
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