Lobesia Botrana Stefanos S

Blackwell Publishing, Ltd. Cold hardiness of diapausing and non-diapausing pupae of the European grapevine moth, Lobesia botrana Stefanos S. Andreadis1, Panagiotis G. Milonas2* & Mathilde Savopoulou-Soultani1 1Laboratory of Applied Zoology and Parasitology, Faculty of Geotechnical Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece, 2Benaki Phytopathological Institute, Department of Entomology, Laboratory of Biological Control, 8 S. Delta St., 14561 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.

early larval stages are exposed to short-day photoperiods Introduction (Deseö et al., 1981). The European grapevine moth, Lobesia botrana (Denis & Insects of the temperate zone adopt a combination of Schiffermüller) (Lepidoptera: Tortricidae), is the most strategies to avoid the damaging effects of exposure to low harmful pest in vineyards of the Mediterranean region. temperature during the winter. At least part of the insect It completes two or three generations per year and populations must be able to tolerate the yearly minimum exceptionally a partial or complete fourth one in the temperature of their overwintering habitats. The majority southern regions (Tzanakakis et al., 1988). Larvae of the of them enter diapause or develop cold hardiness to cope first generation damage the inflorescences and those of with severe winter climates. Diapause and cold hardiness the following generations damage the green, ripening, and are often closely linked in time, but it is not clear how they ripe grape berries. In addition to direct damage, damage to are related, so there is conflicting evidence as to whether ripe or nearly ripe grapes is often accompanied by there is a relation or not (Salt, 1961; Denlinger, 1991; infection of the grapes by the gray mould fungus, Botrytis Hodkova & Hodek, 2004). cinerea Persoon (Sclerotiniaceae) (Savopoulou-Soultani Cold hardiness refers to the capacity of an organism to & Tzanakakis, 1988). Lobesia botrana enters a facultative survive exposure to low temperature and is influenced by autumn hibernal diapause-mediated dormancy in the a variety of factors, including low temperature acclimation pupal stage, which is induced when the embryonic and/or (Lee, 1991; Block, 1995). Acclimation for a few days at low temperatures considerably improved cold hardiness (Nedved, 1995; Ko8tál et al., 1998; Milonas & Savopoulou- *Correspondence: Panagiotis G. Milonas, Benaki Phytopathological Institute, Department of Entomology, Laboratory of Biological Soultani, 1999) although not always (Popham et al., 1991). Control, 8 S. Delta St., 14561 Kifissia, Attikis Greece. In spite of the fact that the biology of L. botrana has been E-mail: [email protected] studied extensively (Deseö et al., 1981; Savopoulou-

Soultani et al., 1998, 1999), studies on the impact of Determination of lethal temperature temperature are limited to the estimation of temperature The temperature, at which 50% and 90% of pupae died, limits for development, and defining day-degrees was determined by cooling groups of 30 individuals required for predicting developmental events in the field (three replicates of 10 pupae for each treatment and sex (Briere & Pracros, 1998; Milonas et al., 2001). separately) to a range of low and subzero temperatures In the present study, we examined the cold tolerance of for 2 h. Exposure temperatures ranged from −3 to −17 °C diapausing and non-diapausing pupae of L. botrana to depending on their physiological condition. Pupae were subzero temperature and determined the supercooling placed in thin-walled test tubes plugged with foam rubber point (SCP) and low lethal temperature (LTemp) of both and immersed in the circulating bath with a solution of physiological stages. We also evaluated the effect of low ethylene glycol and water where the temperature had acclimation in diapausing pupae with respect to their been preset to the desired level. After exposure, non- ability to supercool and to survive at subzero temperature diapausing pupae were transferred to 25 °C under a L16:D8 after a brief exposure at low temperature. 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 Materials and methods pupae were held first at 5 °C under a L8:D16 photoperiod Insects for 50 days following each treatment and then at 10 °C in A laboratory colony of L. botrana was established by continuous darkness for 10 days in order to terminate collecting larvae of different stages from vineyards in diapause. Afterwards, they were transferred to 25 °C under northern Greece (Kavala). Larvae were maintained on a L16:D8 photoperiod where mortality was recorded as for artificial diet (Savopoulou et al., 1994). Adults were placed non-diapausing pupae. in truncated transparent plastic cups covered with tissue paper. A hole was punched at the bottom of each cup and Evaluation of acclimation effect in diapausing pupae was plugged with dental roll wick, which provided the To test if acclimation could enhance survival of diapausing adults with a 5% sucrose solution. The eggs were laid on pupae, groups of pupae (three replicates of 10 pupae the inner walls of the cups and after the removal of adults, for each treatment and sex separately) were placed in pieces of artificial diet were provided for the neonate controlled environmental chambers at 5 °C (Precision larvae. Non-diapausing pupae were obtained by rearing Scientific, General Electric, Louisville, KY, USA) under the insects at 25 °C under a L18:D6 photoperiod. Pupal L8:D16 photoperiod for a period of 6, 12, and 18 days. diapause was induced by rearing larvae at 20 °C under Then they were cooled at −10, −12, −14, and −16 °C for a L8:D16 photoperiod. When larvae approached full 2 h. Mortality was observed the same way as mentioned growth, a strip of corrugated paper was added to each cup before. to provide suitable pupation sites. Paper strips with fully grown larvae or pupae were taken from the cups every Statistical analysis second day and maintained at 25 °C under a L18:D6 Differences between treatment means of SCP were photoperiod. Pupae not developing into adults within compared by t-test and one-way ANOVA (SPSS, 2000). 25 days, although remaining alive, were recorded as Lethal temperatures for 50% and 90% mortality of pupae dormant (Tzanakakis et al., 1988). were calculated by probit analysis after correction for control mortality using Abbott’s formula (Finney, 1952). Determination of supercooling points The effect of acclimation time at 5 °C of diapausing pupae Each pupa (5 days after pupation) was placed individually was estimated using the χ2-test (Sokal & Rohlf, 1995). into a transparent plastic capsule (16 × 7 mm) and immobilized with cotton. A copper constantan Results thermocouple (Digitron 2000T, Kalestead Ltd, UK) was attached to the surface of each pupa to monitor its body Supercooling points temperature. The capsule bearing the pupa with the sensor The mean supercooling point was −22.74 and −22.51 °C was placed in a test tube, which was then immersed in a for non-diapause male and female pupae, respectively. circulating bath (Model 9505, PolyScience, IL, USA) with The respective values of mean supercooling point for a solution of ethylene glycol and water. Cooling rate was set diapausing male and female pupae were −24.83 and at 1 °C min−1. The lowest temperature reached before an −24.53 °C. The difference between diapausing and non- exothermic event that occurred due to release of latent heat diapausing pupae was significant both for male (t = 0.035, was taken as the supercooling point of the individual. P<0.05) and female pupae (t = 0.011, P<0.05). Cold hardiness of Lobesia botrana 115

Table 1 Mean supercooling point (SCP) of diapausing and non-diapausing, male and female pupae of Lobesia botrana

Mean SCP (°C ± SD) [range] Treatment n n Non-diapausing −22.74 ± 0.63aA [−16.8–(–25.1)] 15 −22.51 ± 0.77aA [–17–(–25.5)] 15 Diapausing −24.83 ± 0.28bA [–22.9–(–25.9)] 12 −24.53 ± 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 both sexes. In all cases, the values of LTemp50 and LTemp90 male and female pupae in both treatments, diapausing for females were slightly lower than those for males, (t = 0.575, P<0.05) and non-diapausing (t = 0.822, without being statistically significant. P<0.05), with respect to their supercooling capacity, although values for male pupae were slightly lower. Mean Acclimation effect in diapausing pupae fresh weight of diapausing pupae was 7.33 ± 0.29 and Acclimation of diapausing pupae at 5 °C for various 11.28 ± 0.52 g for male and female pupae, respectively. On periods of time did not have any effect on survival when the other hand, fresh weight for non-diapausing ones was they were exposed to −10 °C for 2 h (Figure 1). At −12 °C, 6.73 ± 0.51 and 9.37 ± 0.72 g for male and female pupae, acclimation had a significant influence on mortality of respectively. Some diapausing pupae supercooled to as low female pupae (χ2 = 4.14, P = 0.035). Non-acclimated as −26 °C. The range of supercooling points for diapausing female pupae suffered 45% mortality whereas, for those pupae was narrower than that for non-diapausing ones. being acclimated for various time periods, mortality However, non-diapause pupae supercooled extensively as ranged between 5% and 15%. When the exposure they did not freeze before temperature in the circulating temperature was reduced to −14 °C and −16 °C, bath reached −16 °C. Acclimation at 5 °C did not have any significant differences were observed between non- significant effect on mean SCP of diapausing pupae for acclimated and acclimated diapausing pupae for both − ° χ2 − ° χ2 both male (F3,39 = 1.465, P = 0.239) and female pupae males ( 14 C: = 15.47, P = 0.0015; 16 C: = 25.72, − ° χ2 (F3,39 = 0.658, P = 0.583) (Table 2). In all cases insects were P = 0.001) and females ( 14 C: = 21.9, P = 0.001; found dead after freezing, which occurred momentarily −16 °C: χ2 = 25.2, P = 0.001). Non-acclimated diapausing when latent heat was released at supercooling point. pupae showed greater mortality than acclimated ones in both sexes. The influence of acclimation on increasing Lethal temperatures cold hardiness of L. botrana pupae was most profound The temperatures that caused 50% and 90% mortality at −16 °C. As the acclimation time lengthened, the differences to diapausing and non-diapausing pupae, for each sex in mortality became more obvious (Figure 1). separately, are shown in Table 3. The LTemp50 of diapausing pupae was significantly lower than that of Discussion non-diapausing pupae for both male and female individuals. No appreciable differences were observed between According to Lee (1991), overwintering insects are divided diapausing and non-diapausing pupae with respect to into two main categories based on their ability to survive

LTemp 90, although diapausing values were slightly lower in 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

Mean SCP (°C ± SD) Treatment n n Non-acclimated −24.83 ± 0.28aA 12 –24.53 ± 0.45aA 12 Acclimated for 6 days −24.38 ± 0.43aA 10 –23.99 ± 0.74aA 10 Acclimated for 12 days −24.54 ± 0.58aA 11 –24.44 ± 0.43aA 9 Acclimated for 18 days −24.41 ± 0.48aA 11 –23.63 ± 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). 116 Andreadis et al.

Table 3 Lethal temperatures (LTemp50 and LTemp90) and conﬁdence limits [95% CL] of diapausing and non-diapausing, male and female Lobesia botrana pupae after exposure to subzero temperatures for 2 h

a a χ2 Sex n d.f. LT50 [95% CL] LT90 [95% CL] P Non-diapausing pupae 240 22 −5.7 [(−6.8) –(−3.9)] −12.3 [(−18.6) –(−10.2)] 17.9 0.710 240 22 −7.4 [(−9.9) –(−5.6)] −14.7 [(−32.0) –(−11.3)] 31.2 0.093 Diapausing pupae 209 19 −12.5 [(−13.1) –(−11.4)] −14.0 [(−15.3) –(−13.4)] 7.6 0.991 210 19 −13.0 [(−13.6) –(−11.7)] −14.9 [(−16.2) –(−14.2)] 16.2 0.643 ain °C.

formation within their tissues are called freeze tolerant above their SCP significantly increased the survival of whereas those that cannot survive ice formation within acclimated diapausing pupae compared to non-acclimated their tissues are called freeze susceptible or freeze intolerant. ones. This result was more obvious as the acclimation time The latter category, which is the largest, consists of species at 5 °C lengthened from 6 days to 18 days, perhaps due to that appear to have a great ability to supercool. Lobesia enhanced accumulation of cryoprotective substances, which botrana presents a further example of insects that avoid was triggered by the parallel increase of the acclimation freezing and have low supercooling points. Although we have period. It is known that low-temperature acclimation is no biochemical data for L. botrana, in other species the strongly associated with the production and accumulation enhanced supercooling capacity is influenced by many factors of cryoprotective substances (Ring, 1982; Sømme, 1982), such as the absence of potential ice-nucleating components especially of glycerol. The fact that acclimation may have or the accumulation of cryoprotectants and other antifreeze influenced survival at non-freezing temperatures while the elements, or both (Sømme, 1982; Lee et al., 1996). A SCP remained unchanged is not uncommon. Kostál et al. cold-hardiness profile similar to that of L. botrana has been (2001) reported some evidence for the cryoprotective role reported for various lepidopterous species that overwinter of accumulated polyols independent of the depression at the pupal stage, such as Pieris brassicae (Pullin & Bale, of SCP. No change was observed with respect to the 1989) and Lacanobia atlantica (Turnock, 1993). supercooling point when Diatraea grandiosella diapausing No significant difference occurred when diapausing laboratory larvae were acclimated at 4 °C for 63 days pupae of L. botrana were acclimated at 5 °C for 6, 12, and (Popham et al., 1991). 18 days, respectively, concerning their ability to supercool. The present study showed that although diapausing However, a 2-h exposure to various subzero temperatures 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 ( )]. Cold hardiness of Lobesia botrana 117 under laboratory conditions for both sexes, few of them Finney DJ (1952) Probit Analysis. Cambridge University Press, could survive extended exposure to −15 °C. Most of them Cambridge, UK. died at subzero temperatures well above their supercooling Hodkova M & Hodek I (2004) Photoperiod, diapause and point. In agreement with many other studies, the cold-hardiness. 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