Entomologia Experimentalis et Applicata 101: 257–263, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Rapid cold hardening in the olive fruit fly Bactrocera oleae under laboratory and field conditions Dimitris S. Koveos Laboratory of Applied Zoology and Parasitology, Aristotle University of Thessaloniki, 540 06 Thessaloniki, Greece (Phone and Fax: +30 31 998845; E-mail: [email protected]) Accepted: October 25, 2001

Key words: Bactrocera oleae, rapid cold hardening, cold shock, olive fruit fly

Abstract A rapid cold hardening response was studied in females and males of the olive fruit fly Bactrocera (Dacus) oleae. When laboratory-reared females and males were transferred and maintained from the rearing temperature of 24 ◦ C for 2 h to −6.5 ◦ C approximately 5% survived. However, conditioning of both females and males for 2 h at various temperatures from 0 to 10 ◦ C before their exposure for 2 h to −6.5 ◦ C increased survival to 80 to 92%. A similar rapid cold hardening response in both females and males was also induced through gradual cooling of the flies at a rate of approximately 0.4 ◦ C per min. The rapid increase in cold tolerance after prior conditioning of the flies to low temperatures, was rapidly lost when they returned to a higher temperature of 24 ◦ C. In the field, in late February and early March, females and males were capable of a rapid cold hardening response. After exposure to the critical temperature they suffered a high mortality when tested in the afternoon and low mortality early in the morning on consecutive days, probably because of differences in the prevailing field temperatures a few hours before testing. This plasticity of cold tolerance gained through rapid cold hardening may allow the flies to survive during periods of the year with great fluctuation in circadian temperatures.

Introduction The olive fruit fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) is a major pest of olives in Mediterranean countries. The fly lays eggs singly in the mesocarp of olive fruits; the larva feeds in the mesocarp and pupates in the fruit or soil. It overwinters as a pupa in the soil or as an adult in the olive grove or other sites (Koveos & Tzanakakis, 1990, and references therein). In coastal northern Greece, during the first half of winter all the preimaginal stages inside the olive fruits and adults in the olive grove are found. Later from mid-January and until summer, only female and male adults are found in the field (Koveos et al., unpubl.). In terrestrial arthropods two general strategies have evolved for survival at low winter temperatures: freeze tolerance and freeze intolerance or avoidance (Lee, 1991; Salt, 1961). Most insects are freeze intolerant and by contrast to freeze tolerant species, ice formation within their tissues is fatal. In freeze in-

tolerant species, the supercooling point (SCP), i.e., the temperature at which body water spontaneously freezes represents the absolute lower lethal temperature. However, several freeze intolerant species are killed or fatally injured after a brief and rapid exposure to low but non-freezing temperatures (Denlinger, 1991; Knight et al., 1986; Lee, 1991; Lee & Denlinger, 1985). The type of non-freezing injury caused by a brief (minutes to a few hours) exposure to low temperature is termed cold shock or direct-chilling injury and occurs in a wide variety of prokaryotic and eukaryotic organisms (Chen et al., 1987; Morris et al., 1983; Watson & Morris, 1987). Cold shock injury could be related to neuromuscular injuries (Kelty et al., 1996). At the cellular level cold shock injury could be related to membrane phase changes (Quinn, 1985), thermoelastic stress (Lee, 1991, and references therein) or damages to critical proteins (Lee et al., 1987). Yet, the mechanism of cold shock injury has not been thoroughly clarified.

258 Cold hardening in preparation for winter is an extensively studied physiological adaptation of many arthropods (Leather et al., 1993; Lee, 1991) activated mainly by extended periods of low temperatures (Baust & Lee, 1982; Lee, 1991) and short photoperiod (Horwath & Duman, 1982). In contrast to overwintering cold hardening, the rapid cold hardening response is induced by either a brief exposure (from minutes to some hours) to low temperatures or through gradual cooling of the experimental animals over a range of temperatures, and results in increased survival after a cold shock exposure (Lee et al., 1987; Kelty & Lee, 1999). The protection accomplished by rapid cold hardening is probably related to the accumulation of cryoprotective substances such as glycerol (Chen et al., 1987). Rapid cold hardening is distinctly different from winter cold hardening since it may occur not only in diapause but also in non-diapause individuals throughout the year (Lee, 1991). Through this process, protection from cold shock injury could be accomplished in late autumn or in early spring when an unusual drop of temperature could cause devastating effects either to actively feeding and reproducing individuals or to dormant overwintering individuals in which winter cold hardening is either not yet fully developed (early winter) or has decreased (early spring). Rapid cold hardening has been studied in several insect species from different orders such as Coleoptera, Diptera, Lepidoptera, Hemiptera, and Thysanoptera (Burks & Hagstrum, 1999; Chen et al., 1987; Coulson & Bale, 1990; 1991; Czajka & Lee, 1990; Kim & Kim, 1997; Larsen & Lee, 1994; Lee, 1991; Lee et al., 1987; McDonald et al., 1997; Nunamaker, 1993; Rosales et al., 1994), as well as in a predatory mite (Broufas & Koveos, 2001). For B. oleae, Fletcher & Zervas (1977) found that acclimation for ten days in a series of temperatures from 5 ◦ to 25 ◦ C affected the torpor temperature (i.e., the temperature at which the flies ceased to reacquire a normal standing position when disturbed during cooling). Torpor temperature of the flies increased with increasing acclimatization temperature. Adults of the tephritid fly Bactrocera (Dacus) tryoni are able to rapidly acclimate with respect to thresholds for torpor and flight (Meats, 1973). However, to our knowledge there are no available data on a rapid cold hardening response in B. oleae. The objectives of this work were (1) to detect the presence of rapid cold hardening response in females and males of B. oleae and determine the temperature

and time needed for its induction, (2) to quantify the rate of cold hardening loss upon the return of the fly to high temperatures and (3) to test whether a rapid cold hardening response occurs naturally in early spring.

Materials and methods Stock colony and experimental flies. The colony of B. oleae used in our work was established from field infested olives collected in the county of Thessaloniki, northern Greece, in early September 1999, i.e., approximately two months before the beginning of the laboratory experiments. Stock females and males were maintained in wooden cages (30 × 30 × 30 cm) in a room with a photoperiod of L16:D8 and a temperature of 24 ◦ C and 60 to 70% r.h. and completed two generations before the beginning of the experiments. For the experiments, stock females oviposited on olive fruit of the variety Megaritiki. After oviposition the infested olives were placed in plastic cups and kept in an incubator at L12:D12 and 20 ◦ C. Pupation took place outside the fruits. The newly formed pupae were collected daily and placed in petri dishes. Emerging females and males were maintained in wooden cages similar to those used for the stock colony at L16:D8 and 24 ◦ C and used in the experiments when ten days old. Stock and experimental flies were provided with a liquid diet containing water, sucrose, and yeast hydrolyzate. In each cage, aproximately 30 females and 30 males were kept. Low temperature tolerance: critical temperature. Ten-day-old females or males were placed in groups of ten in a small cylindrical glass vial (8 × 2 cm) and subsequently immersed in a cool bath. To determine the critical temperature for survival, flies were transferred directly from 24 ◦ C to certain low temperatures in the range from 0 ◦ to −9 ◦ C for 2 h. Subsequently, the flies were maintained at 24 ◦ C and L16:D8 and mortality was assessed after 24 h. We recorded survival as the percentage of females and males able to walk following treatment. Each treatment consisted of four replicates of ten individuals. Control groups of females and males were incubated for 2 h at 24 ◦ C. The highest temperature at which survival drops below 20% was used as a ‘critical temperature’ for the detection of rapid cold hardening. Arcsin–square root transformation of survival percentages were used to equalize variances and two-way analysis of variance

259 with temperature and sex (females or males) as main factors, was performed with the procedure of SPSS 9V. Detection of rapid cold hardening in the laboratory. To determine if a rapid cold hardening response occurs in B. oleae and the conditions of its induction, flies reared at L16:D8 and 24 ◦ C were subjected to the following treatments: (1) They were transferred to temperatures ranging from 0 ◦ C to 25 ◦ C for 2 h or to 5 ◦ C for different periods ranging from 0.5 to 4 h and subsequently exposed for 2 h to the critical low temperature (−6.5 ◦ C). (2) They were gradually cooled at a rate of approximately 0.4 ◦ C per min, from 24 ◦ C to the critical low temperature and maintained at this temperature for 2 h. For each treatment four groups of ten females and ten males were used. Control groups were incubated at 24 ◦ C for the same duration used in the experimental procedure. Survival percentages were Arcsin–square root transformed to equalize variances before performing analysis of variance (ANOVA) with the procedure of SPSS 9V. Duncan multiple range test was applied to test for differences between means (SPSS 9V). Duration of rapid cold hardening. To determine the rate of rapid cold hardening degradation at a higher temperature, groups of females and males preconditioned for 4 h at 5 ◦ C were transferred to 25 ◦ C for a period ranging from 0.25 to 3 h and subsequently exposed for 2 h to the critical temperature of −6.5 ◦ C. For each treatment four groups and the respective controls of ten individuals each were used. Detection of rapid cold hardening in the field. To test whether a rapid cold hardening response could be induced under field conditions, females and males developed as immatures in the laboratory at L12:D12 and 20 ◦ C and maintained as adults in a laboratory room with natural photoperiod and temperature ranging from 15 to 20 ◦ C, when 10 to 15 days old were transferred outside of a northeastern window of our laboratory, on 15 February 2000. Daily fluctuation of field temperature was recorded in a nearby meteorological station. Early in the morning (04:00) and late in the afternoon (16:00) in subsequent dates of late February and early March, flies were transferred from the field cages in groups of ten to cylindrical glass vials (8 × 2 cm) and immersed in the cool bath for 2 h at −7 ◦ C. Afterwards, the flies were maintained in plastic cages at L16:D8 and 24 ◦ C and mortality

Figure 1. Survival (± SE) of adult females (closed bars) and males (open bars) of B. oleae after exposure for 2 h to a series of different low temperatures. Control: flies not exposed to low temperatures. For each treatment four replicates of ten individuals were used. ∗ indicates zero survival. Table 1. Two-way ANOVA on factors (sex and temperature) affecting survival of adults of B. oleae after a 2 h exposure to −6.5 ◦ C Source

Df Mean square F

Sex 1 Temperature 9 Sex × temperature 9 Error 60 Total 80

0.06717 3.669 0.01716 0.0179

P

3.749 0.058 204.756 >0.001 0.958 0.484

was assessed 24 h later. Control groups of ten females and ten males were transferred from the field cages to the laboratory and maintained at L16:D8 and 24 ◦ C without exposure to −7 ◦ C. For each treatment four groups and the respective controls of ten individuals each were used.

Results Critical temperature determination. The survival of adult females and males exposed to temperatures from −9 ◦ C to 0 ◦ C for 2 h is shown in Figure 1. Twoway ANOVA has shown that the effect of temperature on survival is significant whereas the effect of sex and the interaction of sex × temperature are not (Table 1). Almost all the females and males survived after exposure to temperatures from 0 to −4 ◦ C whereas none survived at temperatures below −7 ◦ C. There was a substantial decline in survival over a small range of temperatures from approximately 98% at −4 ◦ C to 5% at −6.5 ◦ C (Figure 1). On the basis of these data we

260

Figure 2. Survival (± SE) of adult females (closed bars) and males (open bars) of B. oleae acclimated for 2 h at a series of different temperatures and subsequently exposed for 2 h to −6.5 ◦ C. For each treatment four replicates of ten individuals were used. Table 2. Two-way ANOVA on factors (sex and temperature of acclimation) affecting survival of adults of B. oleae after a 2 h exposure to −6.5 ◦ C Source

df Mean square F

Sex 1 Temperature 5 Sex × temperature 5 Error 36 Total 48

0.01453 2.48 0.0168 0.07234

Figure 3. Survival (± SE) of adult females (closed bars) and males (open bars) of B. oleae acclimated at 5 ◦ C for different periods and subsequently exposed to the critical temperature (2 h to −6.5 ◦ C). Grad. cool.: gradual cooling from 24 ◦ C to the critical temperature, Contr.: not exposed to low temperature. For each treatment four replicates of ten individuals were used.

P

0.201 0.657 34.287 >0.001 0.161 0.975

choose 2 h at −6.5 ◦ C as the critical temperature for rapid cold hardening detection. Detection of rapid cold hardening in the laboratory. Survival at the critical temperature for 2 h was very high and ranged from 80 to 92% as a result of a previous exposure to 0 ◦ , 5 ◦ and 10 ◦ C for 2 h (Figure 2). By contrast, survival at the critical temperature was significantly lower when the flies were previously exposed to 15 ◦ , 20 ◦ and 25 ◦ C (control) for 2 h. Two way ANOVA has shown that the effect of acclimation temperature had a significant effect on survival, whereas the effect of sex and the interation of sex × acclimation temperature was not significant (Table 2). Survival at the critical temperature for 2 h approached control levels after a previous exposure for 1 h, 2 h, 3 h, and 4 h to 5 ◦ C (Figure 3). However, survival at the critical temperature after 0.5 h at 5 ◦ C was substantially lower (approximately 30%). Gradual cooling of females and males at a rate of 0.4 ◦ C per min to the critical temperature and subsequent exposure to this temperature for 2 h resulted in a substantial increase in survival (40 to 55%) compared with survival of flies that were directly exposed to the critical

Figure 4. Effect of the period of maintenance at 24 ◦ C after prior induction of rapid cold hardening response (4 h at 5 ◦ C) on survival of females (closed bars) and males (open bars) of B. oleae at the critical temperature (2 h at −6.5 ◦ C). For each treatment four replicates of ten individuals were used.

temperature for 2 h (Figure 3). Two way ANOVA has shown that the period of acclimation had a significant effect on survival whereas sex and the interaction sex × period of acclimation had no significant effect on survival (Table 3). Loss of rapid cold hardening response. The protection against cold shock gained through rapid cold hardening at 5 ◦ C was rapidly lost on subsequent maintenance of females and males at 24 ◦ C (Figure 4). Survival after exposure to the critical temperature for 2 h was less than 20% after 0.25 h at 24 ◦ C, and very low to zero and similar to the survival of control groups after 1 to 3 h at 24 ◦ C. Two-way ANOVA on factors affecting the loss of rapid cold hardening has shown that the effect of period of maintenance at 24 ◦ C is significant whereas sex and the interaction sex × period of maintenance are not significant (Table 4).

261 Table 3. Two-way ANOVA on factors (sex and period of acclimation at 5 ◦ C) affecting survival of adults of B. oleae after a 2 h exposure to −6.5 ◦ C Source

df

Mean square

F

P

Sex Period of acclimation Sex × period of acclimation Error Total

1 9 9 48 64

0.009068 1.977 0.02347 0.0689

0.001 28.694 0.341

0.971 >0.001 0.931

Table 4. Two way ANOVA on factors (sex and period of maintenance at 24 ◦ C) affecting survival of rapid cold hardened adults of B. oleae after a 2 h exposure to −6.5 ◦ C Source

df

Mean Square

F

P

Sex Period of maintenance Sex × period of maintenance Error Total

1 5 5 42 56

0.06717 1.295 0.003741 0.01823

3.749 71.032 0.188

0.475 >0.001 0.979

Detection of rapid cold hardening in the field. A substantial variation in cold hardiness of females and males occurs within the same day, following changes in the field daily temperature (Figure 4). More specifically, females maintained in the field and exposed for 2 h to −7 ◦ C at 16:00 of 25 February 2000, suffered a high mortality (72.5%). Mortality at the same temperature was significantly lower (approximately 28%) 12 h later at 4:00 of 26 February, 2000. Still further, 12 h later at 16:00 of 26 February, after exposure to the same temperature mortality percentage was again high (approximately 85%). The gain and loss of rapid cold hardening in field caged females are probably due to the substantial drop and increase of field temperatures during the same day. Males were also found to respond to daily fluctuations of temperature by a gain or loss in cold hardening. After exposure for 2 h to −7 ◦ C mortality percentages of males were very high at 16:00 on two subsequent days (29 February and 1 March, 2000) and substantially lower in the following mornings at 4:00 following a drop in the field temperatures (Figure 5). Control females and males transferred from the field cages to L16:D8 and 24 ◦ C at different times of the day, suffered no mortality after 24 h (not shown in Figure 5).

Discussion Females and males of B. oleae are capable of a rapid cold hardening response, which could be induced either after a short exposure (2 h) to temperatures between 0 and 10 ◦ C or through gradual cooling. Other studies with several insect species have demonstrated that the induction of rapid cold hardening could be accomplished after a brief exposure, even as short as 0.5 h to temperatures between 0 ◦ C and 5 ◦ C (Chen et al., 1987; Czajka & Lee, 1990; Nunamaker, 1993). In B. oleae, a considerable increase in cold hardening was recorded after an 1 h exposure to 5 ◦ C while even after 0.5 h exposure to the same temperature a low but substantial increase was recorded. The rapid cold hardening response in B. oleae could also be induced after cooling at the moderate temperature of 10 ◦ C. To our knowledge, a similar response to 10 ◦ C was reported only for the fruit fly Drososphila melanogaster (Kelty & Lee, 1999) and the predatory mite Euseius finlandicus (Broufas & Koveos, 2001) while for other species studied, 10 ◦ C was too high to induce a rapid cold hardening (Chen et al., 1987; Czajka & Lee, 1990; Nunamaker, 1993). The protection from cold shock gained through rapid cold hardening, for both females and males of B. oleae, was lost within 0.25 h of their return to a

262 and males of B. oleae requires exposure for ten days to temperatures from 5 to 20 ◦ C. In B. tryoni it was found that adults were capable of rapid acclimation with respect to threshold temperatures for torpor and flight (Meats, 1973). In our study we demonstrated that under field conditions in late February and early March in both females and males of B. oleae a substantial increase of cold tolerance was recorded through the night, lost several hours later by the afternoon and gained again next night. This plasticity of cold tolerance in the field probably gained through rapid cold hardening is of critical ecological significance because it may allow the flies to respond quickly and survive the diurnal fluctuations of temperature which occasionally occur in early and late winter and early spring in northern Greece. In the present work the rapid cold hardening response of B. oleae in the field was studied only in late February and early March. Further experiments are now running in our laboratory to study whether this response could be induced during autumn and winter and also to study the effect of rapid cold hardening on subsequent fecundity of females.

Figure 5. Survival (± SE) of females and males of B. oleae transferred from the field to the laboratory at different hours of subsequent dates of late February and early March 2000 and exposed for 2 h to −7 ◦ C. Mortality percentages were determined 24 h later. Open circles represent field temperatures at different times of the day. For each treatment four replicates of ten individuals were used.

higher temperature (25 ◦ C). In other insect species such as Frankliniella occidentalis and Musca domestica 1 h at a high temperature could result in the complete loss of cold tolerance gained through rapid cold hardening (Coulson & Bale, 1990; McDonald et al., 1997). In several insect species rapid cold hardening in the laboratory could be induced through gradual cooling (Chen et al., 1987; Coulson & Bale, 1990; McDonald et al., 1997; Kelty & Lee, 1999). In most of the studied cases rapid cold hardening was accomplished at cooling rates of approximately 0.5 per min. In our study only one cooling rate (0.4 ◦ C per min) was tested and resulted in a considerable increase in cold hardening. Under field conditions in northern Greece, circadian changes of field temperatures experienced by the flies are not so abrupt and as found in our experiments favor a rapid cold hardening response (Figure 5). Previous work by Fletcher & Zervas (1977) has shown that acclimation to low temperatures of females

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