Cold hardiness of diapausing and non-diapausing pupae of the European grapevine moth, Lobesia botrana

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1 Blackwell Publishing, Ltd. Cold hardiness of diapausing and non-diapausing pupae of the European grapevine moth, Lobesia botrana Stefanos S. Andreadis 1, Panagiotis G. Milonas 2 * & Mathilde Savopoulou-Soultani 1 1 Laboratory of Applied Zoology and Parasitology, Faculty of Geotechnical Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece, 2 Benaki Phytopathological Institute, Department of Entomology, Laboratory of Biological Control, 8 S. Delta St., Kifissia, Attikis Greece Accepted: 9 June 2005 Key words: supercooling point, acclimation, prefreezing mortality, Lepidoptera, Tortricidae Abstract Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae) is a key pest of grapes in Europe. It overwinters as a pupa in the bark crevices of the plant. Supercooling point (SCP) and low temperature survival was investigated in the laboratory and was determined using a cool bath and a 1 C min 1 cooling rate. Freezing was fatal both to diapausing and non-diapausing pupae. SCP was significantly lower in diapausing male ( 24.8 C) and female ( 24.5 C) pupae than in non-diapausing ones ( 22.7 and 22.5 C, respectively). Sex had no influence on SCP both for diapausing and non-diapausing pupae. Supercooling was also not affected by acclimation. However, acclimation did improve survival of diapausing pupae at temperatures above the SCP. Survival increased as acclimation period increased and the influence was more profound at the lower temperatures examined. Diapausing pupae could withstand lower temperatures than non-diapausing ones and lethal temperature was significantly lower than for non-diapausing pupae. Freezing injury above the SCP has been well documented for both physiological stages of L. botrana pupae. Our findings suggest a diapause-related cold hardiness for L. botrana and given its cold hardiness ability, winter mortality due to low temperatures is not expected to occur, especially in southern Europe. Introduction The European grapevine moth, Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae), is the most harmful pest in vineyards of the Mediterranean region. It completes two or three generations per year and exceptionally a partial or complete fourth one in the southern regions (Tzanakakis et al., 1988). Larvae of the first generation damage the inflorescences and those of the following generations damage the green, ripening, and ripe grape berries. In addition to direct damage, damage to ripe or nearly ripe grapes is often accompanied by infection of the grapes by the gray mould fungus, Botrytis cinerea Persoon (Sclerotiniaceae) (Savopoulou-Soultani & Tzanakakis, 1988). Lobesia botrana enters a facultative autumn hibernal diapause-mediated dormancy in the pupal stage, which is induced when the embryonic and/or *Correspondence: Panagiotis G. Milonas, Benaki Phytopathological Institute, Department of Entomology, Laboratory of Biological Control, 8 S. Delta St., Kifissia, Attikis Greece. biocon@bpi.gr early larval stages are exposed to short-day photoperiods (Deseö et al., 1981). Insects of the temperate zone adopt a combination of strategies to avoid the damaging effects of exposure to low temperature during the winter. At least part of the insect populations must be able to tolerate the yearly minimum temperature of their overwintering habitats. The majority of them enter diapause or develop cold hardiness to cope with severe winter climates. Diapause and cold hardiness are often closely linked in time, but it is not clear how they are related, so there is conflicting evidence as to whether there is a relation or not (Salt, 1961; Denlinger, 1991; Hodkova & Hodek, 2004). Cold hardiness refers to the capacity of an organism to survive exposure to low temperature and is influenced by a variety of factors, including low temperature acclimation (Lee, 1991; Block, 1995). Acclimation for a few days at low temperatures considerably improved cold hardiness (Nedved, 1995; Ko8tál et al., 1998; Milonas & Savopoulou- Soultani, 1999) although not always (Popham et al., 1991). In spite of the fact that the biology of L. botrana has been studied extensively (Deseö et al., 1981; Savopoulou The Netherlands Entomological Society Entomologia Experimentalis et Applicata 117: ,

2 114 Andreadis et al. Soultani et al., 1998, 1999), studies on the impact of temperature are limited to the estimation of temperature limits for development, and defining day-degrees required for predicting developmental events in the field (Briere & Pracros, 1998; Milonas et al., 2001). In the present study, we examined the cold tolerance of diapausing and non-diapausing pupae of L. botrana to subzero temperature and determined the supercooling point (SCP) and low lethal temperature (LTemp) of both physiological stages. We also evaluated the effect of low acclimation in diapausing pupae with respect to their ability to supercool and to survive at subzero temperature after a brief exposure at low temperature. Materials and methods Insects A laboratory colony of L. botrana was established by collecting larvae of different stages from vineyards in northern Greece (Kavala). Larvae were maintained on artificial diet (Savopoulou et al., 1994). Adults were placed in truncated transparent plastic cups covered with tissue paper. A hole was punched at the bottom of each cup and was plugged with dental roll wick, which provided the adults with a 5% sucrose solution. The eggs were laid on the inner walls of the cups and after the removal of adults, pieces of artificial diet were provided for the neonate larvae. Non-diapausing pupae were obtained by rearing the insects at 25 C under a L18:D6 photoperiod. Pupal diapause was induced by rearing larvae at 20 C under a L8:D16 photoperiod. When larvae approached full growth, a strip of corrugated paper was added to each cup to provide suitable pupation sites. Paper strips with fully grown larvae or pupae were taken from the cups every second day and maintained at 25 C under a L18:D6 photoperiod. Pupae not developing into adults within 25 days, although remaining alive, were recorded as dormant (Tzanakakis et al., 1988). Determination of supercooling points Each pupa (5 days after pupation) was placed individually into a transparent plastic capsule (16 7 mm) and immobilized with cotton. A copper constantan thermocouple (Digitron 2000T, Kalestead Ltd, UK) was attached to the surface of each pupa to monitor its body temperature. The capsule bearing the pupa with the sensor was placed in a test tube, which was then immersed in a circulating bath (Model 9505, PolyScience, IL, USA) with a solution of ethylene glycol and water. Cooling rate was set at 1 C min 1. The lowest temperature reached before an exothermic event that occurred due to release of latent heat was taken as the supercooling point of the individual. Determination of lethal temperature The temperature, at which 50% and 90% of pupae died, was determined by cooling groups of 30 individuals (three replicates of 10 pupae for each treatment and sex separately) to a range of low and subzero temperatures for 2 h. Exposure temperatures ranged from 3 to 17 C depending on their physiological condition. Pupae were placed in thin-walled test tubes plugged with foam rubber and immersed in the circulating bath with a solution of ethylene glycol and water where the temperature had been preset to the desired level. After exposure, nondiapausing pupae were transferred to 25 C under a L16:D8 photoperiod. Pupae were considered dead if they did not emerge after 30 days or if they had visible signs of deformation after emergence. In contrast, diapausing pupae were held first at 5 C under a L8:D16 photoperiod for 50 days following each treatment and then at 10 C in continuous darkness for 10 days in order to terminate diapause. Afterwards, they were transferred to 25 C under a L16:D8 photoperiod where mortality was recorded as for non-diapausing pupae. Evaluation of acclimation effect in diapausing pupae To test if acclimation could enhance survival of diapausing pupae, groups of pupae (three replicates of 10 pupae for each treatment and sex separately) were placed in controlled environmental chambers at 5 C (Precision Scientific, General Electric, Louisville, KY, USA) under L8:D16 photoperiod for a period of 6, 12, and 18 days. Then they were cooled at 10, 12, 14, and 16 C for 2 h. Mortality was observed the same way as mentioned before. Statistical analysis Differences between treatment means of SCP were compared by t-test and one-way ANOVA (SPSS, 2000). Lethal temperatures for 50% and 90% mortality of pupae were calculated by probit analysis after correction for control mortality using Abbott s formula (Finney, 1952). The effect of acclimation time at 5 C of diapausing pupae was estimated using the χ 2 -test (Sokal & Rohlf, 1995). Results Supercooling points The mean supercooling point was and C for non-diapause male and female pupae, respectively. The respective values of mean supercooling point for diapausing male and female pupae were and C. The difference between diapausing and nondiapausing pupae was significant both for male (t = 0.035, P<0.05) and female pupae (t = 0.011, P<0.05).

3 Cold hardiness of Lobesia botrana 115 Table 1 Mean supercooling point (SCP) of diapausing and non-diapausing, male and female pupae of Lobesia botrana Treatment Mean SCP ( C ± SD) [range] n n Non-diapausing ± 0.63aA [ 16.8 ( 25.1)] ± 0.77aA [ 17 ( 25.5)] 15 Diapausing ± 0.28bA [ 22.9 ( 25.9)] ± 0.45bA [ 20.8 ( 26.3)] 12 Means followed by the same lower case letter within a column and by the same capital letter within a line are not significantly different (t-test; P<0.05). (Table 1). No appreciable difference was observed between male and female pupae in both treatments, diapausing (t = 0.575, P<0.05) and non-diapausing (t = 0.822, P<0.05), with respect to their supercooling capacity, although values for male pupae were slightly lower. Mean fresh weight of diapausing pupae was 7.33 ± 0.29 and ± 0.52 g for male and female pupae, respectively. On the other hand, fresh weight for non-diapausing ones was 6.73 ± 0.51 and 9.37 ± 0.72 g for male and female pupae, respectively. Some diapausing pupae supercooled to as low as 26 C. The range of supercooling points for diapausing pupae was narrower than that for non-diapausing ones. However, non-diapause pupae supercooled extensively as they did not freeze before temperature in the circulating bath reached 16 C. Acclimation at 5 C did not have any significant effect on mean SCP of diapausing pupae for both male (F 3,39 = 1.465, P = 0.239) and female pupae (F 3,39 = 0.658, P = 0.583) (Table 2). In all cases insects were found dead after freezing, which occurred momentarily when latent heat was released at supercooling point. Lethal temperatures The temperatures that caused 50% and 90% mortality to diapausing and non-diapausing pupae, for each sex separately, are shown in Table 3. The LTemp 50 of diapausing pupae was significantly lower than that of non-diapausing pupae for both male and female individuals. No appreciable differences were observed between diapausing and non-diapausing pupae with respect to LTemp 90, although diapausing values were slightly lower in both sexes. In all cases, the values of LTemp 50 and LTemp 90 for females were slightly lower than those for males, without being statistically significant. Acclimation effect in diapausing pupae Acclimation of diapausing pupae at 5 C for various periods of time did not have any effect on survival when they were exposed to 10 C for 2 h (Figure 1). At 12 C, acclimation had a significant influence on mortality of female pupae (χ 2 = 4.14, P = 0.035). Non-acclimated female pupae suffered 45% mortality whereas, for those being acclimated for various time periods, mortality ranged between 5% and 15%. When the exposure temperature was reduced to 14 C and 16 C, significant differences were observed between nonacclimated and acclimated diapausing pupae for both males ( 14 C: χ 2 = 15.47, P = ; 16 C: χ 2 = 25.72, P = 0.001) and females ( 14 C: χ 2 = 21.9, P = 0.001; 16 C: χ 2 = 25.2, P = 0.001). Non-acclimated diapausing pupae showed greater mortality than acclimated ones in both sexes. The influence of acclimation on increasing cold hardiness of L. botrana pupae was most profound at 16 C. As the acclimation time lengthened, the differences in mortality became more obvious (Figure 1). Discussion According to Lee (1991), overwintering insects are divided into two main categories based on their ability to survive the freezing of their body water. Insects that can survive ice Table 2 Mean supercooling point (SCP) of diapausing, non-acclimated and acclimated Lobesia botrana pupae at 5 C for 6, 12, and 18 days Treatment Mean SCP ( C ± SD) n n Non-acclimated ± 0.28aA ± 0.45aA 12 Acclimated for 6 days ± 0.43aA ± 0.74aA 10 Acclimated for 12 days ± 0.58aA ± 0.43aA 9 Acclimated for 18 days ± 0.48aA ± 0.52aA 10 Means followed by the same lower case letter within a column and by the same capital letter within a line are not significantly different (Fisher Protected LSD test, P<0.05).

4 116 Andreadis et al. Table 3 Lethal temperatures (LTemp 50 and LTemp 90 ) and confidence limits [95% CL] of diapausing and non-diapausing, male and female Lobesia botrana pupae after exposure to subzero temperatures for 2 h Sex n d.f. LT 50 a [95% CL] LT 90 a [95% CL] χ 2 P Non-diapausing pupae [( 6.8) ( 3.9)] 12.3 [( 18.6) ( 10.2)] [( 9.9) ( 5.6)] 14.7 [( 32.0) ( 11.3)] Diapausing pupae [( 13.1) ( 11.4)] 14.0 [( 15.3) ( 13.4)] [( 13.6) ( 11.7)] 14.9 [( 16.2) ( 14.2)] a in C. formation within their tissues are called freeze tolerant whereas those that cannot survive ice formation within their tissues are called freeze susceptible or freeze intolerant. The latter category, which is the largest, consists of species that appear to have a great ability to supercool. Lobesia botrana presents a further example of insects that avoid freezing and have low supercooling points. Although we have no biochemical data for L. botrana, in other species the enhanced supercooling capacity is influenced by many factors such as the absence of potential ice-nucleating components or the accumulation of cryoprotectants and other antifreeze elements, or both (Sømme, 1982; Lee et al., 1996). A cold-hardiness profile similar to that of L. botrana has been reported for various lepidopterous species that overwinter at the pupal stage, such as Pieris brassicae (Pullin & Bale, 1989) and Lacanobia atlantica (Turnock, 1993). No significant difference occurred when diapausing pupae of L. botrana were acclimated at 5 C for 6, 12, and 18 days, respectively, concerning their ability to supercool. However, a 2-h exposure to various subzero temperatures above their SCP significantly increased the survival of acclimated diapausing pupae compared to non-acclimated ones. This result was more obvious as the acclimation time at 5 C lengthened from 6 days to 18 days, perhaps due to enhanced accumulation of cryoprotective substances, which was triggered by the parallel increase of the acclimation period. It is known that low-temperature acclimation is strongly associated with the production and accumulation of cryoprotective substances (Ring, 1982; Sømme, 1982), especially of glycerol. The fact that acclimation may have influenced survival at non-freezing temperatures while the SCP remained unchanged is not uncommon. Kostál et al. (2001) reported some evidence for the cryoprotective role of accumulated polyols independent of the depression of SCP. No change was observed with respect to the supercooling point when Diatraea grandiosella diapausing laboratory larvae were acclimated at 4 C for 63 days (Popham et al., 1991). The present study showed that although diapausing pupae of L. botrana could be supercooled below 24 C Figure 1 Mortality ± SE of Lobesia botrana male and female diapausing pupae subjected to four subzero temperatures after various periods of acclimation at 5 C [diapausing pupae ( ), diapausing pupae acclimated for 6 days ( ), diapausing pupae acclimated for 12 days ( ), diapausing pupae acclimated for 18 days ( )].

5 Cold hardiness of Lobesia botrana 117 under laboratory conditions for both sexes, few of them could survive extended exposure to 15 C. Most of them died at subzero temperatures well above their supercooling point. In agreement with many other studies, the supercooling point is not sufficient to describe the dynamic process of insect cold hardiness, leading to the conclusion that non-freeze injury is as fatal as freezing. For both physiological stages, the lower lethal temperature limits for survival probably occur well above the supercooling point for most pupae. As Bale (1987) concluded in a review of insect cold hardiness, from an ecological standpoint, the determination of supercooling capacity is necessary but not always sufficient for characterizing the limit of cold tolerance. Laboratory diapausing pupae of L. botrana had a significantly lower mean SCP than non-diapausing pupae (5 days after pupation). Furthermore, the lethal temperature of diapausing pupae, which is needed to cause 50% mortality (LTemp 50 ) in a population, was significantly lower than that of non-diapausing pupae for both sexes, indicating the existence of a strong relation between cold hardiness and diapause (Denlinger, 1991). Such a difference in cold tolerance between diapausing and non-diapausing states has been confirmed for another tortricid species, Adoxophyes orana (Milonas & Savopoulou-Soultani, 1999). The exact parameters that regulate the process are not yet known and more detailed experiments are needed in order to verify the correctness of our findings. Although we acknowledge that in our experiments we have not included naturally occurring conditions, our findings nonetheless suggest that winter mortality of diapausing pupae due to freezing is not likely to occur in northern Greece because temperatures seldom fall below 10 C and not for more than a few hours (Milonas & Savopoulou-Soultani, 1999). 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