Seasonal Changes in the Cold Hardiness of the Two-Spotted Spider Mite Females (Acari: Tetranychidae) Author(s): S. Khodayari, H. Colinet, S. Moharramipour, and D. Renault Source: Environmental Entomology, 42(6):1415-1421. Published By: Entomological Society of America URL: http://www.bioone.org/doi/full/10.1603/EN13086
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PHYSIOLOGICAL ECOLOGY
Seasonal Changes in the Cold Hardiness of the Two-Spotted Spider Mite Females (Acari: Tetranychidae) S. KHODAYARI,1 H. COLINET,2 S. MOHARRAMIPOUR,1,3
AND
D. RENAULT2
Environ. Entomol. 42(6): 1415Ð1421 (2013); DOI: http://dx.doi.org/10.1603/EN13086
ABSTRACT The twospotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) is an important agricultural pest. Population dynamics and pest outbreaks highly depend on the overwintering success of the mite specimens; therefore, it is necessary to assess winter survival dynamics of this pest. Seasonal changes in supercooling point (SCP) and acute cold tolerance (2-h exposure at ⫺5, ⫺10, ⫺15, ⫺20, ⫺23, or ⫺25⬚C) were assessed in Þeld-collected females during the winter in 2010 Ð2011 in Iran. The SCP values varied from a minimum of ⫺30.5⬚C (January 2011) to a maximum of ⫺12.6⬚C (April 2011). SigniÞcant differences were recorded in the SCP distribution patterns between autumnand winter-sampled females, depicting the acquisition of cold hardiness over the winter. The mean ambient air temperature was the lowest in January (4⬚C), when the females showed the highest supercooling ability. Correlated patterns between monthly temperatures and acute cold tolerance also were found. At ⫺20⬚C, the survival of the mites was very low (10%) when they were sampled in October 2010; whereas it was high (97.5%) in January 2011, before decreasing to 5% in April 2011. The present data show that T. urticae females are chill tolerant and capable of adjusting their cold tolerance over the winter season. Acute cold tolerance (⫺15 and ⫺20⬚C) and SCP represent valuable metrics that can be used for predicting the seasonal changes of the cold hardiness of T. urticae females. KEY WORDS winter survival, supercooling point, lethal temperature, pest, Acari
In temperate and polar regions, arthropods may endure prolonged exposures at subzero temperatures, which they can survive in one of two ways (Salt 1961, Voituron et al. 2002, Denlinger and Lee 2010): First, freeze-tolerant arthropods can withstand extracellular ice formation by actively inducing ice nucleation at high subzero temperatures (intracellular freezing is lethal in arthropods; Sinclair and Renault 2010). Second, freeze-intolerant (or freeze-avoiding) arthropods cannot survive freezing. To survive in a supercooled state, freeze-intolerant species show an array of physiological and biochemical adjustments to seasonally increase their supercooling ability, and thus reduce freezing risks (Zachariassen 1985, Storey and Storey 1992). In these species, the supercooling point (SCP) corresponds to the temperature at which body ßuids freeze, and thus represents the lowest thermal limit that a freeze-intolerant species can handle (Bale 1987). Even if the ecological relevance of the SCP has been disputed (Renault et al. 2002), it can constitute a valid metrics of the cold tolerance in some insect species (Worland and Convey 2001, Klok et al. 2003). SCP frequency distributions often are characterized by a range of different patterns, from multimodal to normal 1 Department of Entomology, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran. 2 Universite ´ de Rennes 1, UMR CNRS 6553 Ecobio, 263 avenue du Gal Leclerc, 35042 Rennes, France. 3 Corresponding author, e-mail: [email protected].
distributions, reßecting for instance the seasonal changes in cold tolerance via changes in the feeding and physiological status (for instance molting and diapause), body water hydration (cryoprotective dehydration), and cold acclimation (Sømme and Block 1982, Knight et al. 1986, Salin et al. 2000, Colinet et al. 2007, Verdu´ 2011). In addition to SCP, the critical thermal limits and the lethal time, or the lethal temperature, for 50% of the population (Lt50 and LT50, respectively) also represent valuable metrics that can be used to assess the cold hardiness of arthropod species (Bale 1987, 1993; Turnock and Fields 2005; Renault 2011). The twospotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), whose genome has recently been sequenced (Grbic et al. 2011), are distributed worldwide and represent a pest affecting a large range of agricultural crops (van de Vrie et al. 1972, van Leeuwen et al. 2012). T. urticae overwinters as mated females in reproductive diapause in the soil of infested Þelds (Plant and Wilson 1985). This reproductive diapause is induced by a combination of environmental factors, including short photoperiod, low temperature, and unfavorable food supply (Veerman 1977, Kawakami et al. 2009, Ito et al. 2013). Diapause usually terminates in the spring in T. urticae, and is associated with an increase of the cold hardiness of the females (Khodayari et al. 2013). As a result of the severe proliferation of this pest species worldwide, most studies conducted on T. urticae so far have fo-
0046-225X/13/1415Ð1421$04.00/0 䉷 2013 Entomological Society of America
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Fig. 1. Daily minimum, maximum, and average air temperatures recorded at the Chitgar agrometeorological station (Tehran, Iran) from October 2010 to April 2011. Arrows represent the sampling dates of female T. urticae.
cused on their biological and chemical control (Lilley and Campbell 1999, Opit et al. 2004, van Leeuwen et al. 2010, Dermauw et al. 2012), whereas little is known about their cold tolerance during winter periods (Khodayari et al. 2012, 2013). Finally, the spider mites represent some of the major arthropod crop pests, but seasonal studies of the overwintering survival of this species in either Þeld or greenhouse are lacking. In the present work, we aimed at describing the seasonal changes in the cold hardiness of the twospotted spider mite in Iran (semiarid continental climate), where T. urticae Þrst was observed in 1954 (Davatchi and Taghizadeh 1954). As reported previously by Danks (2005), it is important to couple Þeld and laboratory experiments because experiments designed without natural environmental data may lack ecological relevance. Thus, female T. urticae were Þeld-sampled at regular intervals throughout the winter period in 2010 Ð2011, and their cold tolerance was directly assessed by measuring their supercooling ability and survival to acute cold stress. The current study provides the Þrst information on the cold tolerance of the overwintering females of T. urticae from this region of the world. Materials and Methods Mites. Adult females of T. urticae were hand-collected monthly in the Þeld from ornamental cabbage Brassica oleracea L. at Tarbiat Modares University (35⬚ 44⬘N, 51⬚ 10⬘E, Tehran, Iran) from October 2010 to April 2011. The course of the temperature change (air temperature) during the overwintering period is shown in Fig. 1. Outdoor climatic data were obtained from the Chitgar agrometeorological station, which is 1 km away from the sampling site. The cabbage leaves harboring mites were placed in plastic bags, and, within the few minutes after collection, they were immediately transferred to the laboratory, and directly subjected to cold tolerance assays. Supercooling Ability. The SCP of adult females was determined by attaching the tip of a copper-constan-
tan thermocouple to the dorsal idiosoma of each adult female with a small spot of glue. SCP was measured in 14 Ð17 individuals for each sampling date (seven sampling dates, see Fig. 1). We used a programmable refrigerated test chamber (model MK 57, Binder GmbH Bergstr, Tuttlingen, Germany) in combination with temperature data logger (model 177-T4, Testo, Lenzkirch, Germany) that transferred the data at 2-s intervals into a computer. The data were read using the Comsoft 3.0 software. The temperature was cooled from outdoor temperatures to the SCP at a constant cooling rate of 0.5⬚C/min. The SCP was recorded at the start of the exotherm produced by the latent heat of freezing. Survival to Acute Cold Stress. The change in cold hardiness of females over the cold season in 2010 Ð2011 was assessed by measuring survival to a range of acute cold exposures for each sampling date. Females were placed in a temperature controlled chamber, where the temperature was lowered from 20⬚C to ⫺5, ⫺10, ⫺15, ⫺20, ⫺23, or ⫺25⬚C at a rate of 1⬚C/min. Four to Þve replicates, each consisting of a pool of 10 females, were used for each of the six endpoint temperatures. The cooling was stopped when the desired temperature was reached, and the temperature was maintained constant over the next 2 h. Then, the temperature was raised to 20⬚C at a rate of 1⬚C/min, and all mites were allowed to recover for 1 d at room temperature and then checked for survival. As the females of T. urticae remain active during their reproductive diapause, mortality was scored as the number of mites that exhibited no movements. Statistical Analysis. The normality of all SCP data were Þrst checked using the ShapiroÐWilk statistic (␣ ⫽ 0.05). In most cases, the SCP data failed to Þt normality (P ⬍ 0.05) and sometimes tended to show a form of bimodal distribution. Hence, analyses based on comparison of the distributional patterns were used because statistical tests based on mean values are not relevant in this case (Worland et al. 2006). Thus, KolmogorovÐSmirnov (KÐS) two-sample tests were used to compare the SCP distributions (Worland et al.
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2006). For the acute cold tolerance, survival curves along with their 95% conÞdence intervals (CI) for the proportion were Þrst dressed. Then, the temperatureÐ mortality regression equations were calculated using binary logistic regressions to determine the lethal temperatures for 50 (LT50) and 90% (LT90) of the population and their 95 and 99% CIs (upper and lower limits). Correlations between acute cold tolerance data and mean monthly temperatures were checked using Spearman tests. All statistical analyses were conducted using MINITAB Statistical Software Release 13 (MINITAB Inc., State College, PA) and SPSS version 16.0 (Chicago). Results Meteorological Data. Daily maximum, minimum, and average air temperatures recorded from 1 October 2010 to 30 April 2011 are shown in Fig. 1. The mean air temperature was 23.6 ⫾ 0.4⬚C in October 2010, 4.0 ⫾ 0.5⬚C in January 2011, and 15.6 ⫾ 0.6⬚C in April 2011. The total number of freezing days (number of days below 0⬚C) was 32 d during the sampling period (December 2010: 1 d; January 2011: 11 d; February 2011: 14 d; and March 2011: 6 d), with a minimum temperature of ⫺6.6⬚C recorded in January 2011. Supercooling Ability. Adult females of T. urticae showed a marked seasonal variability in their supercooling abilities (Fig. 2). The SCP values ranged from ⫺30.5⬚C (the lowest SCP value recorded) in January 2011 to ⫺12.6⬚C (the highest SCP value recorded) in April 2011. The distribution pattern of the SCP values did not differ among the sampling dates during the period OctoberÐDecember 2010 (Fig. 2; P ⬎ 0.05). Then, the SCP distributions signiÞcantly differed between December 2010 and January 2011 (KÐS ⫽ 0.6071, P ⬍ 0.01), with median SCP values of ⫺23.2 and ⫺26.3⬚C (i.e., a shift toward lower SCP values), respectively. The distribution of SCP values also differed from February to March 2011 (KÐS ⫽ 0.4706, P ⬍ 0.05). Finally, the SCP distribution in March 2011 was signiÞcantly different from that which has been measured in April 2011 (KÐS ⫽ 0.6078, P ⬍ 0.001; i.e., a shift toward higher SCP values; Fig. 2). Survival to Acute Cold Stress. Adult females of T. urticae also showed seasonal variability in their ability to survive acute cold stress for 2 h (Fig. 3). A significant correlation was found between the survival to exposures at ⫺15 and ⫺20⬚C for 2 h and the mean monthly temperatures (r ⫽ ⫺0.852, P ⬍ 0.05 and r ⫽ ⫺0.964, P ⬍ 0.01, respectively), whereas the other experimental temperatures were not correlated with the mean monthly temperatures (P ⬎ 0.05). At ⫺20⬚C, the survival of the mites was very low (10%) in October 2010; it was very high (97.5%) in January 2011 before declining to 5% in April 2011. None of the female T. urticae sampled in October, November, December, and April survived the 2 h-exposure at ⫺23⬚C. Based on binary logistic regressions, LT50 and LT90 were estimated for each sampling date (Fig. 4). LT50 was signiÞcantly correlated with the mean monthly temperatures (r ⫽ 0.929, P ⬍ 0.01), whereas no cor-
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relation was observed for LT90 (r ⫽ 0.714, P ⫽ 0.088). LT50 was the lowest in January, February, and March 2011 (ca. ⫺22.5⬚C, P ⬍ 0.01), and the highest in April 2011 (⫺11.1 ⫾ 0.4⬚C, P ⬍ 0.01). LT50 and LT90 significantly differed (P ⬍ 0.05) among the sampling dates (Fig. 4).
Discussion The current study examined the cold tolerance of Þeld-collected T. urticae females during the winter period in 2010 Ð2011 in Tehran, Iran. The SCP values decreased from ca. ⫺23 to ca. ⫺26⬚C with decreasing ambient temperature (from an average of 23⬚C in October 2010 to 4⬚C in January 2011) and then increased. The few published studies that examined the supercooling ability of T. urticae reported that body ßuids of this mite freeze at ⫺18.7⬚C in overwintering females collected from greenhouses in Norway (Stenseth 1965). Nondiapausing females also seem to be characterized by higher SCP values (ca. ⫺19⬚C) than diapausing ones (ca. ⫺24⬚C) (Khodayari et al. 2012). The present SCP values measured from Þeldsampled specimens are congruent with these early observations. These SCP values are also similar with those reported from other oribatid mites, whose SCP ranges from ⫺20 to ⫺30⬚C in winter-acclimated individuals (Schatz and Sømme 1981, Block and Sømme 1982). SigniÞcant differences were recorded in the SCP distribution between autumn- and winter-sampled females, depicting the acquisition of cold hardiness over the winter. A mixture of nondiapausing and diapausing mites is usually observed in autumn in the Þeld. The relative abundance of diapausing females over nondiapausing ones increased from 38.9% in October to 97.7% in February (Klingen et al. 2008). From January to March 2011, overwintering females, which exhibited the lowest SCP, were most likely diapausing. The diapausing status of T. urticae females was not monitored in the present work, but it is already known that the SCP values differ between diapausing and nondiapausing phenotypes, with diapausing specimens freezing at lower temperatures (Khodayari et al. 2012). Mites stop feeding when entering diapause in early winter, and this cessation of feeding may enhance supercooling abilities through the evacuation of the gut and the elimination of indigenous ice nucleators (Block et al. 1978, Watanabe and Tanaka 1998, Ramløv 2000, Colinet et al. 2007). Moreover, diapause is usually accompanied by a range of physiological and biochemical adjustments (Colinet et al. 2012), which subsequently increase the cold hardiness of the organisms (Denlinger 1991, Atapour and Moharramipour 2009). The accumulation of “winter” polyols (inositol, mannitol, and sorbitol) in overwintering females may also account for a colligative depression of their SCP (Khodayari et al. 2013). The dramatic increase in SCP in April 2011 may be associated with termination of diapause. In this period, a wide range of SCP values was observed, which might reßect a
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Fig. 2. Frequency distribution of the supercooling points of female T. urticae sampled in the Þeld from October 2010 to April 2011. Median SCP values (⫾average absolute deviation) are presented for each month. Results from the two-sample KÐS tests are indicated for each distribution comparison.
mixed population of diapausing and nondiapausing phenotypes. We also monitored the seasonal change in T. urticae cold tolerance by exposing monthly collected specimens to a range of acute cold stresses and demonstrated that the mites became cold hardy in winter. Of note, the mortality rates of the specimens exposed at ⫺5 and ⫺10⬚C for 2 h were low and did not change signiÞcantly among the sampling periods. The high survival rate at these thermal conditions suggests that the mites of this region are chill tolerant according to BaleÕs classiÞcation (Bale 2002). This view is reinforced by the fact that female mites were even able to
survive at temperatures ranging from ⫺15 to ⫺23⬚C that were close to their SCP values. Chill tolerant species are characterized by extensive supercooling abilities and a high level of cold tolerance as opposed to chill susceptible species, which do not tolerate exposures to subzero temperatures for prolonged periods of time (Bale 2002). The chill tolerance of T. urticae may be considered as a component of its worldwide invasive success. Further studies should also conduct chronic cold tolerance assays because the duration of cold exposure is as important as the temperature per se. In addition, because the genome of T. urticae has recently been sequenced, annotated,
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Fig. 3. Survival rate (⫾95% CI for the proportion) of T. urticae females collected in the Þeld from October 2010 to April 2011 and exposed at ⫺5, ⫺10, ⫺15, ⫺20, ⫺23, or ⫺25⬚C for 2 h.
and published (Grbic et al. 2011), there is enormous potential to use this model organism to better understand the cold hardiness of other mite species. The seasonal variation of the cold hardiness of T. urticae, well pictured by the SCP values over the period October 2010 ÐApril 2011, was conÞrmed by the survival ability of the mites exposed to acute cold stress. During the coldest months of the year, the mites were characterized by the lowest LT50 values. Moreover, LT50 values were highly correlated with seasonal temperatures, and among all experimental temperatures used to assess LT50, we found that ⫺20⬚C (for 2 h) best represented the seasonal variation of the cold hardiness of the mites. Indeed, the percentage of mites surviving at this experimental condition varied from ⬇10% in October, November, and April to nearly 100% during the coldest months of the year (January and February). At ⫺25⬚C (for 2 h), a temperature that was very close to the median SCP of the overwintering mites, almost no survival was observed. This study shows that T. urticae females are chill tolerant and are able to increase their cold tolerance toward the winter season. In addition, we found that SCP and LT50 represent valuable metrics for the monitoring of the seasonal changes of the cold hardiness of
this pest species. Our study suggests that overwintering females of T. urticae exposed to cold and frosty nights during the winter are unlikely to experience substantial mortality from cold conditions alone in Iran. Meanwhile, their overwintering success also depends on the combination of many other abiotic and biotic variables that should be further examined, such as the duration and frequency of cold events, loss of homeostatic control, exhaustion of the body reserves, body dehydration, or pathogens. We also suggest that the survival ability of female T. urticae to chronic cold exposure or repeated frost events should be considered in further studies. Finally, to improve the management practices and to predict pest outbreaks, we need to characterize the overwintering strategies employed by pest species. In this context, the current study provides valuable basic information on the cold survival of the spider mite over the winter season. Acknowledgments We thank the Tarbiat Modares University (Tehran, Iran), which funded the experiments and also the travel of S. Khodayari from Iran to France. The work in Iran was funded by the Tarbiat Modares University grants to S. Moharramipour.
Fig. 4. LT50 and LT90 values with 95% lower and upper CIs of Þeld-collected females of T. urticae from October 2010 to April 2011. Distinct letters among the sampling dates for LT50 and LT90 represent the statistical differences among the values according to the 95% CI.
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This research was supported by the University of Rennes one (Action Incitative de Recherche 2012ÑCollaborations Internationales) and the Centre National de la Recherche (CNRS). We would also like to thank the anonymous referees for their helpful comments on an earlier version of this paper.
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Verdu´ , J. R. 2011. Chill tolerance variability within and among populations in the dung beetle Canthon humectus hidalgoensis along an altitudinal gradient in the Mexican semiarid high plateau. J. Arid Environ. 75: 119 Ð124. Voituron, Y., N. Mouquet, C. deMazancourt, and J. Clobert. 2002. To freeze or not to freeze? An evolutionary perspective on the cold hardiness strategies of overwintering ectotherms. Am. Nat. 160: 255Ð270. Watanabe, M., and K. Tanaka. 1998. Adult diapause and cold hardiness in Aulacophora nigripennis (Coleoptera: Chrysomelidae). J. Insect Physiol. 44: 1103Ð1110. Worland, M. R., and P. Convey. 2001. Rapid cold hardening in Antarctic microarthropods. Funct. Ecol. 15: 515Ð524. Worland, M. R., H. P. Leinaas, and S. L. Chown. 2006. Supercooling point frequency distributions in Collembola are affected by moulting. Funct. Ecol. 20: 323Ð329. Zachariassen, K. E. 1985. Physiology of cold tolerance in insects. Physiol. Rev. 65: 799 Ð 832. Received 29 March 2013; accepted 8 October 2013.
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PHYSIOLOGICAL ECOLOGY
Seasonal Changes in the Cold Hardiness of the Two-Spotted Spider Mite Females (Acari: Tetranychidae) S. KHODAYARI,1 H. COLINET,2 S. MOHARRAMIPOUR,1,3
AND
D. RENAULT2
Environ. Entomol. 42(6): 1415Ð1421 (2013); DOI: http://dx.doi.org/10.1603/EN13086
ABSTRACT The twospotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) is an important agricultural pest. Population dynamics and pest outbreaks highly depend on the overwintering success of the mite specimens; therefore, it is necessary to assess winter survival dynamics of this pest. Seasonal changes in supercooling point (SCP) and acute cold tolerance (2-h exposure at ⫺5, ⫺10, ⫺15, ⫺20, ⫺23, or ⫺25⬚C) were assessed in Þeld-collected females during the winter in 2010 Ð2011 in Iran. The SCP values varied from a minimum of ⫺30.5⬚C (January 2011) to a maximum of ⫺12.6⬚C (April 2011). SigniÞcant differences were recorded in the SCP distribution patterns between autumnand winter-sampled females, depicting the acquisition of cold hardiness over the winter. The mean ambient air temperature was the lowest in January (4⬚C), when the females showed the highest supercooling ability. Correlated patterns between monthly temperatures and acute cold tolerance also were found. At ⫺20⬚C, the survival of the mites was very low (10%) when they were sampled in October 2010; whereas it was high (97.5%) in January 2011, before decreasing to 5% in April 2011. The present data show that T. urticae females are chill tolerant and capable of adjusting their cold tolerance over the winter season. Acute cold tolerance (⫺15 and ⫺20⬚C) and SCP represent valuable metrics that can be used for predicting the seasonal changes of the cold hardiness of T. urticae females. KEY WORDS winter survival, supercooling point, lethal temperature, pest, Acari
In temperate and polar regions, arthropods may endure prolonged exposures at subzero temperatures, which they can survive in one of two ways (Salt 1961, Voituron et al. 2002, Denlinger and Lee 2010): First, freeze-tolerant arthropods can withstand extracellular ice formation by actively inducing ice nucleation at high subzero temperatures (intracellular freezing is lethal in arthropods; Sinclair and Renault 2010). Second, freeze-intolerant (or freeze-avoiding) arthropods cannot survive freezing. To survive in a supercooled state, freeze-intolerant species show an array of physiological and biochemical adjustments to seasonally increase their supercooling ability, and thus reduce freezing risks (Zachariassen 1985, Storey and Storey 1992). In these species, the supercooling point (SCP) corresponds to the temperature at which body ßuids freeze, and thus represents the lowest thermal limit that a freeze-intolerant species can handle (Bale 1987). Even if the ecological relevance of the SCP has been disputed (Renault et al. 2002), it can constitute a valid metrics of the cold tolerance in some insect species (Worland and Convey 2001, Klok et al. 2003). SCP frequency distributions often are characterized by a range of different patterns, from multimodal to normal 1 Department of Entomology, Faculty of Agriculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran. 2 Universite ´ de Rennes 1, UMR CNRS 6553 Ecobio, 263 avenue du Gal Leclerc, 35042 Rennes, France. 3 Corresponding author, e-mail: [email protected].
distributions, reßecting for instance the seasonal changes in cold tolerance via changes in the feeding and physiological status (for instance molting and diapause), body water hydration (cryoprotective dehydration), and cold acclimation (Sømme and Block 1982, Knight et al. 1986, Salin et al. 2000, Colinet et al. 2007, Verdu´ 2011). In addition to SCP, the critical thermal limits and the lethal time, or the lethal temperature, for 50% of the population (Lt50 and LT50, respectively) also represent valuable metrics that can be used to assess the cold hardiness of arthropod species (Bale 1987, 1993; Turnock and Fields 2005; Renault 2011). The twospotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), whose genome has recently been sequenced (Grbic et al. 2011), are distributed worldwide and represent a pest affecting a large range of agricultural crops (van de Vrie et al. 1972, van Leeuwen et al. 2012). T. urticae overwinters as mated females in reproductive diapause in the soil of infested Þelds (Plant and Wilson 1985). This reproductive diapause is induced by a combination of environmental factors, including short photoperiod, low temperature, and unfavorable food supply (Veerman 1977, Kawakami et al. 2009, Ito et al. 2013). Diapause usually terminates in the spring in T. urticae, and is associated with an increase of the cold hardiness of the females (Khodayari et al. 2013). As a result of the severe proliferation of this pest species worldwide, most studies conducted on T. urticae so far have fo-
0046-225X/13/1415Ð1421$04.00/0 䉷 2013 Entomological Society of America
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Fig. 1. Daily minimum, maximum, and average air temperatures recorded at the Chitgar agrometeorological station (Tehran, Iran) from October 2010 to April 2011. Arrows represent the sampling dates of female T. urticae.
cused on their biological and chemical control (Lilley and Campbell 1999, Opit et al. 2004, van Leeuwen et al. 2010, Dermauw et al. 2012), whereas little is known about their cold tolerance during winter periods (Khodayari et al. 2012, 2013). Finally, the spider mites represent some of the major arthropod crop pests, but seasonal studies of the overwintering survival of this species in either Þeld or greenhouse are lacking. In the present work, we aimed at describing the seasonal changes in the cold hardiness of the twospotted spider mite in Iran (semiarid continental climate), where T. urticae Þrst was observed in 1954 (Davatchi and Taghizadeh 1954). As reported previously by Danks (2005), it is important to couple Þeld and laboratory experiments because experiments designed without natural environmental data may lack ecological relevance. Thus, female T. urticae were Þeld-sampled at regular intervals throughout the winter period in 2010 Ð2011, and their cold tolerance was directly assessed by measuring their supercooling ability and survival to acute cold stress. The current study provides the Þrst information on the cold tolerance of the overwintering females of T. urticae from this region of the world. Materials and Methods Mites. Adult females of T. urticae were hand-collected monthly in the Þeld from ornamental cabbage Brassica oleracea L. at Tarbiat Modares University (35⬚ 44⬘N, 51⬚ 10⬘E, Tehran, Iran) from October 2010 to April 2011. The course of the temperature change (air temperature) during the overwintering period is shown in Fig. 1. Outdoor climatic data were obtained from the Chitgar agrometeorological station, which is 1 km away from the sampling site. The cabbage leaves harboring mites were placed in plastic bags, and, within the few minutes after collection, they were immediately transferred to the laboratory, and directly subjected to cold tolerance assays. Supercooling Ability. The SCP of adult females was determined by attaching the tip of a copper-constan-
tan thermocouple to the dorsal idiosoma of each adult female with a small spot of glue. SCP was measured in 14 Ð17 individuals for each sampling date (seven sampling dates, see Fig. 1). We used a programmable refrigerated test chamber (model MK 57, Binder GmbH Bergstr, Tuttlingen, Germany) in combination with temperature data logger (model 177-T4, Testo, Lenzkirch, Germany) that transferred the data at 2-s intervals into a computer. The data were read using the Comsoft 3.0 software. The temperature was cooled from outdoor temperatures to the SCP at a constant cooling rate of 0.5⬚C/min. The SCP was recorded at the start of the exotherm produced by the latent heat of freezing. Survival to Acute Cold Stress. The change in cold hardiness of females over the cold season in 2010 Ð2011 was assessed by measuring survival to a range of acute cold exposures for each sampling date. Females were placed in a temperature controlled chamber, where the temperature was lowered from 20⬚C to ⫺5, ⫺10, ⫺15, ⫺20, ⫺23, or ⫺25⬚C at a rate of 1⬚C/min. Four to Þve replicates, each consisting of a pool of 10 females, were used for each of the six endpoint temperatures. The cooling was stopped when the desired temperature was reached, and the temperature was maintained constant over the next 2 h. Then, the temperature was raised to 20⬚C at a rate of 1⬚C/min, and all mites were allowed to recover for 1 d at room temperature and then checked for survival. As the females of T. urticae remain active during their reproductive diapause, mortality was scored as the number of mites that exhibited no movements. Statistical Analysis. The normality of all SCP data were Þrst checked using the ShapiroÐWilk statistic (␣ ⫽ 0.05). In most cases, the SCP data failed to Þt normality (P ⬍ 0.05) and sometimes tended to show a form of bimodal distribution. Hence, analyses based on comparison of the distributional patterns were used because statistical tests based on mean values are not relevant in this case (Worland et al. 2006). Thus, KolmogorovÐSmirnov (KÐS) two-sample tests were used to compare the SCP distributions (Worland et al.
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2006). For the acute cold tolerance, survival curves along with their 95% conÞdence intervals (CI) for the proportion were Þrst dressed. Then, the temperatureÐ mortality regression equations were calculated using binary logistic regressions to determine the lethal temperatures for 50 (LT50) and 90% (LT90) of the population and their 95 and 99% CIs (upper and lower limits). Correlations between acute cold tolerance data and mean monthly temperatures were checked using Spearman tests. All statistical analyses were conducted using MINITAB Statistical Software Release 13 (MINITAB Inc., State College, PA) and SPSS version 16.0 (Chicago). Results Meteorological Data. Daily maximum, minimum, and average air temperatures recorded from 1 October 2010 to 30 April 2011 are shown in Fig. 1. The mean air temperature was 23.6 ⫾ 0.4⬚C in October 2010, 4.0 ⫾ 0.5⬚C in January 2011, and 15.6 ⫾ 0.6⬚C in April 2011. The total number of freezing days (number of days below 0⬚C) was 32 d during the sampling period (December 2010: 1 d; January 2011: 11 d; February 2011: 14 d; and March 2011: 6 d), with a minimum temperature of ⫺6.6⬚C recorded in January 2011. Supercooling Ability. Adult females of T. urticae showed a marked seasonal variability in their supercooling abilities (Fig. 2). The SCP values ranged from ⫺30.5⬚C (the lowest SCP value recorded) in January 2011 to ⫺12.6⬚C (the highest SCP value recorded) in April 2011. The distribution pattern of the SCP values did not differ among the sampling dates during the period OctoberÐDecember 2010 (Fig. 2; P ⬎ 0.05). Then, the SCP distributions signiÞcantly differed between December 2010 and January 2011 (KÐS ⫽ 0.6071, P ⬍ 0.01), with median SCP values of ⫺23.2 and ⫺26.3⬚C (i.e., a shift toward lower SCP values), respectively. The distribution of SCP values also differed from February to March 2011 (KÐS ⫽ 0.4706, P ⬍ 0.05). Finally, the SCP distribution in March 2011 was signiÞcantly different from that which has been measured in April 2011 (KÐS ⫽ 0.6078, P ⬍ 0.001; i.e., a shift toward higher SCP values; Fig. 2). Survival to Acute Cold Stress. Adult females of T. urticae also showed seasonal variability in their ability to survive acute cold stress for 2 h (Fig. 3). A significant correlation was found between the survival to exposures at ⫺15 and ⫺20⬚C for 2 h and the mean monthly temperatures (r ⫽ ⫺0.852, P ⬍ 0.05 and r ⫽ ⫺0.964, P ⬍ 0.01, respectively), whereas the other experimental temperatures were not correlated with the mean monthly temperatures (P ⬎ 0.05). At ⫺20⬚C, the survival of the mites was very low (10%) in October 2010; it was very high (97.5%) in January 2011 before declining to 5% in April 2011. None of the female T. urticae sampled in October, November, December, and April survived the 2 h-exposure at ⫺23⬚C. Based on binary logistic regressions, LT50 and LT90 were estimated for each sampling date (Fig. 4). LT50 was signiÞcantly correlated with the mean monthly temperatures (r ⫽ 0.929, P ⬍ 0.01), whereas no cor-
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relation was observed for LT90 (r ⫽ 0.714, P ⫽ 0.088). LT50 was the lowest in January, February, and March 2011 (ca. ⫺22.5⬚C, P ⬍ 0.01), and the highest in April 2011 (⫺11.1 ⫾ 0.4⬚C, P ⬍ 0.01). LT50 and LT90 significantly differed (P ⬍ 0.05) among the sampling dates (Fig. 4).
Discussion The current study examined the cold tolerance of Þeld-collected T. urticae females during the winter period in 2010 Ð2011 in Tehran, Iran. The SCP values decreased from ca. ⫺23 to ca. ⫺26⬚C with decreasing ambient temperature (from an average of 23⬚C in October 2010 to 4⬚C in January 2011) and then increased. The few published studies that examined the supercooling ability of T. urticae reported that body ßuids of this mite freeze at ⫺18.7⬚C in overwintering females collected from greenhouses in Norway (Stenseth 1965). Nondiapausing females also seem to be characterized by higher SCP values (ca. ⫺19⬚C) than diapausing ones (ca. ⫺24⬚C) (Khodayari et al. 2012). The present SCP values measured from Þeldsampled specimens are congruent with these early observations. These SCP values are also similar with those reported from other oribatid mites, whose SCP ranges from ⫺20 to ⫺30⬚C in winter-acclimated individuals (Schatz and Sømme 1981, Block and Sømme 1982). SigniÞcant differences were recorded in the SCP distribution between autumn- and winter-sampled females, depicting the acquisition of cold hardiness over the winter. A mixture of nondiapausing and diapausing mites is usually observed in autumn in the Þeld. The relative abundance of diapausing females over nondiapausing ones increased from 38.9% in October to 97.7% in February (Klingen et al. 2008). From January to March 2011, overwintering females, which exhibited the lowest SCP, were most likely diapausing. The diapausing status of T. urticae females was not monitored in the present work, but it is already known that the SCP values differ between diapausing and nondiapausing phenotypes, with diapausing specimens freezing at lower temperatures (Khodayari et al. 2012). Mites stop feeding when entering diapause in early winter, and this cessation of feeding may enhance supercooling abilities through the evacuation of the gut and the elimination of indigenous ice nucleators (Block et al. 1978, Watanabe and Tanaka 1998, Ramløv 2000, Colinet et al. 2007). Moreover, diapause is usually accompanied by a range of physiological and biochemical adjustments (Colinet et al. 2012), which subsequently increase the cold hardiness of the organisms (Denlinger 1991, Atapour and Moharramipour 2009). The accumulation of “winter” polyols (inositol, mannitol, and sorbitol) in overwintering females may also account for a colligative depression of their SCP (Khodayari et al. 2013). The dramatic increase in SCP in April 2011 may be associated with termination of diapause. In this period, a wide range of SCP values was observed, which might reßect a
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Fig. 2. Frequency distribution of the supercooling points of female T. urticae sampled in the Þeld from October 2010 to April 2011. Median SCP values (⫾average absolute deviation) are presented for each month. Results from the two-sample KÐS tests are indicated for each distribution comparison.
mixed population of diapausing and nondiapausing phenotypes. We also monitored the seasonal change in T. urticae cold tolerance by exposing monthly collected specimens to a range of acute cold stresses and demonstrated that the mites became cold hardy in winter. Of note, the mortality rates of the specimens exposed at ⫺5 and ⫺10⬚C for 2 h were low and did not change signiÞcantly among the sampling periods. The high survival rate at these thermal conditions suggests that the mites of this region are chill tolerant according to BaleÕs classiÞcation (Bale 2002). This view is reinforced by the fact that female mites were even able to
survive at temperatures ranging from ⫺15 to ⫺23⬚C that were close to their SCP values. Chill tolerant species are characterized by extensive supercooling abilities and a high level of cold tolerance as opposed to chill susceptible species, which do not tolerate exposures to subzero temperatures for prolonged periods of time (Bale 2002). The chill tolerance of T. urticae may be considered as a component of its worldwide invasive success. Further studies should also conduct chronic cold tolerance assays because the duration of cold exposure is as important as the temperature per se. In addition, because the genome of T. urticae has recently been sequenced, annotated,
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Fig. 3. Survival rate (⫾95% CI for the proportion) of T. urticae females collected in the Þeld from October 2010 to April 2011 and exposed at ⫺5, ⫺10, ⫺15, ⫺20, ⫺23, or ⫺25⬚C for 2 h.
and published (Grbic et al. 2011), there is enormous potential to use this model organism to better understand the cold hardiness of other mite species. The seasonal variation of the cold hardiness of T. urticae, well pictured by the SCP values over the period October 2010 ÐApril 2011, was conÞrmed by the survival ability of the mites exposed to acute cold stress. During the coldest months of the year, the mites were characterized by the lowest LT50 values. Moreover, LT50 values were highly correlated with seasonal temperatures, and among all experimental temperatures used to assess LT50, we found that ⫺20⬚C (for 2 h) best represented the seasonal variation of the cold hardiness of the mites. Indeed, the percentage of mites surviving at this experimental condition varied from ⬇10% in October, November, and April to nearly 100% during the coldest months of the year (January and February). At ⫺25⬚C (for 2 h), a temperature that was very close to the median SCP of the overwintering mites, almost no survival was observed. This study shows that T. urticae females are chill tolerant and are able to increase their cold tolerance toward the winter season. In addition, we found that SCP and LT50 represent valuable metrics for the monitoring of the seasonal changes of the cold hardiness of
this pest species. Our study suggests that overwintering females of T. urticae exposed to cold and frosty nights during the winter are unlikely to experience substantial mortality from cold conditions alone in Iran. Meanwhile, their overwintering success also depends on the combination of many other abiotic and biotic variables that should be further examined, such as the duration and frequency of cold events, loss of homeostatic control, exhaustion of the body reserves, body dehydration, or pathogens. We also suggest that the survival ability of female T. urticae to chronic cold exposure or repeated frost events should be considered in further studies. Finally, to improve the management practices and to predict pest outbreaks, we need to characterize the overwintering strategies employed by pest species. In this context, the current study provides valuable basic information on the cold survival of the spider mite over the winter season. Acknowledgments We thank the Tarbiat Modares University (Tehran, Iran), which funded the experiments and also the travel of S. Khodayari from Iran to France. The work in Iran was funded by the Tarbiat Modares University grants to S. Moharramipour.
Fig. 4. LT50 and LT90 values with 95% lower and upper CIs of Þeld-collected females of T. urticae from October 2010 to April 2011. Distinct letters among the sampling dates for LT50 and LT90 represent the statistical differences among the values according to the 95% CI.
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This research was supported by the University of Rennes one (Action Incitative de Recherche 2012ÑCollaborations Internationales) and the Centre National de la Recherche (CNRS). We would also like to thank the anonymous referees for their helpful comments on an earlier version of this paper.
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Khodayari, S., S. Moharramipour, K. Kamali, M. Jalali Javaran, and D. Renault. 2012. Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae. J. Therm. Biol. 37: 419 Ð 423. Khodayari, S., S. Moharramipour, V. Larvor, K. Hidalgo, and D. Renault. 2013. Metabolic changes associated with cold-hardiness in the two-spotted spider mite: effect of acclimation and diapause. PLoS ONE 8: e54025. Klingen, I., G. Wærsted, and K. Westrum. 2008. Overwintering and prevalence of Neozygites floridana (Zygomycetes: Neozygitaceae) in hibernating females of Tetranychus urticae (Acari: Tetranychidae) under cold climatic conditions in strawberries. Exp. Appl. Acarol. 46: 231Ð245. Klok, C. J., S. L. Chown, and K. J. Gaston. 2003. The geographic ranges structure of the holly leaf-miner. III. Cold hardiness physiology. Funct. Ecol. 17: 858 Ð 868. Knight, J. D., J. S. Bale, F. Franks, S. F. Mathias, and J. G. Baust. 1986. Insect cold hardiness: supercooling points and pre-freeze mortality. Cryo Letters 7: 194 Ð203. Lilley, R., and C.A.M. Campbell. 1999. Biological, chemical and integrated control of two-spotted spider mite Tetranychus urticae on dwarf hops. Biocontrol Sci. Technol. 9: 467Ð 473. Opit, G. P., J. R. Nechols, and D. C. Margolies. 2004. Biological control of twospotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), using Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseidae) on ivy geranium: assessment of predator release ratios. Biol. Control 29: 445Ð 452. Plant, R. E., and L. T. Wilson. 1985. A bayesian method for sequential sampling and forecasting in agricultural pest management. Biometrics 4: 203Ð214. Ramløv, H. 2000. Aspects of natural cold tolerance in ectothermic animals. Hum. Reprod. 15: 26 Ð 46. Renault, D. 2011. Long-term after-effects of cold exposure in adult Alphitobius diaperinus (Tenebrionidae): the need to link survival ability with subsequent reproductive success. Ecol. Entomol. 36: 36 Ð 42. Renault, D., C. Salin, G. Vannier, and P. Vernon. 2002. Survival at low temperatures in insects: what is the ecological signiÞcance of the supercooling point? Cryo Letters 23: 217Ð228. Salin, C., D. Renault, G. Vannier, and P. Vernon. 2000. As sexually dimorphic response in supercooling temperature, enhanced by starvation, in the lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae). J. Therm. Biol. 25: 411Ð 418. Salt, R. W. 1961. Principles of insect cold hardiness. Annu. Rev. Entomol. 6: 55Ð74. Schatz, H., and L. Sømme. 1981. Cold-hardiness of some oribatid mites from the Alps. Cryo Letters 2: 207Ð216. Sinclair, B., and D. Renault. 2010. Intracellular ice formation in insects: unresolved after 50 years? Comp. Biochem. Physiol. A 155: 14 Ð18. Sømme, L., and W. Block. 1982. Cold hardiness of Collembola at Signy Island, maritime Antarctic. Oikos 38: 168 Ð 176. Stenseth, C. 1965. Cold hardiness in the two-spotted spider mite (Tetranychus urticae Koch). Entomol. Exp. Appl. 8: 33Ð38. Storey, K. B., and J. M. Storey. 1992. Biochemical adaptations for winter survival in insects, pp. 101Ð140. In P. L. Steponkus (ed.), Advances in low-temperature biology. JAI Press, London, United Kingdom.
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