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UV tolerance in the two-spotted spider mite, Tetranychus urticae
Journal of Insect Physiology 55 (2009) 649–654
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UV tolerance in the two-spotted spider mite, Tetranychus urticae Takeshi Suzuki a,*, Masakatsu Watanabe b,c, Makio Takeda a a
Graduate School of Agricultural Science, Kobe University, Rokko-dai, Nada, Kobe 657-8501, Japan National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 444-8585, Japan c Department of Photoscience, School of Advanced Sciences, Graduate University for Advanced Studies, Shonan Village, Hayama, Kanagawa 240-0193, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2009 Received in revised form 14 April 2009 Accepted 14 April 2009
The two-spotted spider mite, Tetranychus urticae was exposed to UV-C (250 nm), UV-B (300 nm), and UV-A (350 nm). In non-diapausing females, the median effective doses for 50% mortality plus escape incidence (ED50) were 21 (UV-C) and 104 kJ m2 (UV-B); those for 50% oviposition rate in continuous darkness-treated mites were 6.2 (UV-C) and 41 kJ m2 (UV-B). No significant effects of UV-A on mortality and oviposition rate were observed. The ED50 values for UV-B were similar to the natural UV-B observed for 2–5 days in summer when T. urticae inhabits the undersides of leaves. Therefore, T. urticae possibly uses leaves as a filter to avoid the deleterious effects of UV-B. In diapausing females, low mortality was observed even at high doses of UV radiation, but more than half escaped even at low doses. The orange body color of diapausing females results from accumulation of carotenoids, a scavenger for UV-induced reactive oxygen species; this may explain the low mortality of diapausing females. Diapausing females may overcome the deleterious effects of UV-B during winter in the absence of leaves by emigrating to UV-free environments and by accumulating carotenoids. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: UV irradiation Diapause Pest control Phototaxis Tetranychus urticae
1. Introduction Ultraviolet (UV) radiation directly damages DNA and is absorbed by some coenzymes and pigments in vivo; UV absorption raises these compounds to an excited state, and the excitation energy is finally transferred to H2O molecules, yielding reactive oxygen species (ROS). It has been suggested that UV-induced ROS damages important intra- and extracellular components, such as lipids, lipid membranes, nucleic acids, and proteins (Jurkiewicz and Buettner, 1994; Shindo et al., 1994). Such UV/ROS-induced damage would be lethal to mites because of their small size; i.e., their body surface area per weight is large. Therefore, artificial UV irradiation could prove to be a promising non-chemical measure for reducing populations of the two-spotted spider mite (Tetranychus urticae), which is distributed worldwide and causes serious damage to a wide variety of crops (van de Vrie et al., 1972). There is abundant information on the response to light, particularly photoperiodism, in T. urticae. Only adult females enter diapause in order to survive during winter by sensing longnight conditions, and diapausing females, which exhibit a orange body color in this period, do not feed or oviposit (Veerman, 1985). Response to UV radiation in T. urticae has been investigated with regard to behavior (McEnroe and Dronka, 1966; Naegele et al.,
* Corresponding author. Tel.: +81 78 803 5870; fax: +81 78 803 5870. E-mail address: [email protected] (T. Suzuki). 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.04.005
1966; Barcelo and Calkins, 1980; Barcelo, 1981), tolerance (Barcelo, 1981), and light-dependent enzyme activity (Suzuki et al., 2008b, 2009). However, these experiments were conducted with non-diapausing females, and data on the response of diapausing females to UV radiation is limited. The present study investigates differences in tolerance to UV radiation between non-diapausing and diapausing adult females and discusses phenological adaptations to UV radiation in T. urticae. 2. Materials and methods 2.1. Laboratory culture of T. urticae The founder population of T. urticae was collected from an apple (Malus pumila Mill. cv. Fuji) orchard located in Akita, Japan (398150 N), in 2001. The offspring population was maintained in the laboratory on kidney bean leaves (Phaseolus vulgaris L.) under longday (LD 16:8) conditions provided by white fluorescent lamps at 25 8C. The adult females that emerged from the teleiochrysalis stage within 3 days were used in the following UV tolerance experiment. To obtain diapausing females, adult females were placed on a fresh kidney bean leaf that was positioned on water-soaked cotton in a plastic Petri dish (diameter, 9 cm; depth, 2 cm); they were kept there 12 h under continuous darkness (DD) at 25 8C for oviposition and were removed after eggs were laid. The eggs were maintained for 5 days under the same environmental conditions; then, the
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larvae that hatched were exposed to short-day (LD 8:16) conditions provided by white light-emitting diodes (LEDs) (NNN28300; Panasonic Electric Works Co. Ltd., Osaka, Japan) at 18 8C. Diapause induction was determined 7–10 days after adult emergence, and the adult females with uniform orange coloration were used in the following UV tolerance experiment. The nondiapausing and diapausing females were then placed on fresh leaf disks (diameter, 1 cm; 1 individual per disk) kept over watersoaked cotton in the plastic Petri dish. 2.2. UV irradiation The effects of UV radiation on the mortality, escape, and oviposition rates in T. urticae were determined by using the Okazaki Large Spectrograph (Watanabe et al., 1982) at the National Institute for Basic Biology, Okazaki, Japan. Monochromatic light was provided by a large spectrograph equipped with a 30 kW xenon arc lamp (Ushio Electric Co., Tokyo, Japan). The light beam was first reflected by a plane mirror and then by a condensing mirror. After reflection by a diffraction grating, the light beam passed into the irradiation boxes. In each Petri dish placed in the boxes with radiation wavelengths of 250, 300, and 350 nm, T. urticae samples were placed beneath a mirror that reflected the monochromatic light beam directly onto them. Table 1 lists the types of treatment, peak wavelength, irradiance, and dosages. Fig. 1 shows the spectral distributions of the treatments when irradiance ranging from 200 to 800 nm was set at 100 W m2. The irradiance values (0.06–0.6 W m2 for UV-C and 0.2– 2.4 W m2 for UV-B and UV-A) at specific wavelengths were
adjusted with UV filters (Fujitok Co., Tokyo, Japan) and measured with a radiation sensor (QTM-101; Monotech Inc., Saitama, Japan). Samples were treated under a light–dark cycle (LD 4:20) with monochromatic light of different irradiances for 3 days. 2.3. Observations After daily irradiation, the numbers of non-diapausing and diapausing females that were alive, dead, or had escaped, and the number of eggs laid by non-diapausing females were observed with a stereomicroscope (SZ-PT; Olympus Co., Tokyo, Japan). Viability or mortality was judged with the help of a brush; individuals that showed no reaction on contact with the brush were regarded as dead. Individuals that had fallen off the leaf disk onto the water-soaked cotton were considered to have escaped. The observation was conducted under the red light (>600 nm) that T. urticae could not sense (Suzuki et al., 2008a); thus, the undesirable effects of light were prevented. 2.4. Statistical analysis Statistical significances of differences between mortality and escape incidence under DD (control) and those exposed to different irradiances at a specific wavelength were determined by Fisher’s exact test. The statistical significance of the differences between the oviposition rate under DD and those exposed to different irradiances at a specific wavelength was determined by one-way ANOVA and then Dunnett’s test. A correlation analysis was conducted to determine the UV dose response in terms of mortality, escape, and oviposition. The calculations were performed using SigmaPlot 2001 (SPSS Inc., Chicago, IL) and SPSS 11.5J software (SPSS Japan Inc., Tokyo, Japan). 3. Results 3.1. Mortality In non-diapausing females, differences between mortality in all UV treatments and those in the control conditions (DD) were not significant on days 1 and 2 (P > 0.05, Fisher’s exact test); the mortality was nearly 0% (Table 2). Even on day 3, mortality in the UV-C, UV-B, and UV-A treatments with irradiances below 0.2, 0.6, and 2.4 W m2, respectively, was not significantly different from those under DD (P > 0.05). However, mortality on day 3 was
Fig. 1. Spectral distributions of the irradiation treatments when the irradiance was set at 100 W m2.
Table 2 Daily changes in mortality in non-diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Irradiance [W m2]
Na
Cumulative mortality [%] Day 1
Table 1 Type of UV radiation, peak wavelength, irradiance, and daily-integrated dose.
Day 3
Control (DD)
–
98
1
2
4
UV-C
0.06 0.2 0.6
60 65 65
0NS 0NS 0NS
0NS 3NS 3NS
7NS 6NS 18**
0.9 2.9 8.6
UV-B
0.2 0.6 2.4
60 59 60
0NS 2NS 0NS
2NS 2NS 3NS
3NS 2NS 32***
UV-A
0.2 0.6 2.4
60 60 60
0NS 0NS 0NS
0NS 2NS 0NS
0NS 5NS 0NS
Type of UV radiation
Peak wavelength [nm]
Irradiance [W m2]
UV-C
250
0.06 0.2 0.6
UV-B
300
0.2 0.6 2.4
2.9 8.6 34.6
UV-A
350
0.2 0.6 2.4
2.9 8.6 34.6
The exposure time was 4 h per day.
Day 2
Daily-integrated dose [kJ m2 day1]
a
Number of individuals. P < 0.01. P < 0.001, NS: not significant (P > 0.05) between the control and UV treatments within the same column by Fisher’s exact test, DD: continuous darkness. **
***
T. Suzuki et al. / Journal of Insect Physiology 55 (2009) 649–654 Table 3 Daily changes in mortality in diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Irradiance [W m2]
Na
Day 1
Day 2
Table 5 Daily changes in escape percentage in diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Cumulative mortality [%]
651
Day 3
Irradiance [W m2]
Na
Cumulative escape [%] Day 1
Day 2
Day 3
Control (DD)
–
72
0
1
1
Control (DD)
–
72
3
8
10
UV-C
0.06 0.2 0.6
52 57 65
6NS 2NS 0NS
10NS 4NS 0NS
10NS 5NS 6NS
UV-C
0.06 0.2 0.6
52 57 65
19*** 21*** 14***
35*** 35*** 42***
38** 40*** 51***
UV-B
0.2 0.6 2.4
50 52 60
0NS 0NS 0NS
4NS 2NS 3NS
6NS 6NS 5NS
UV-B
0.2 0.6 2.4
50 52 60
38*** 27*** 28***
68*** 42*** 52***
68*** 44*** 63***
UV-A
0.2 0.6 2.4
50 50 59
0NS 2NS 0NS
2NS 8NS 3NS
2NS 8NS 8NS
UV-A
0.2 0.6 2.4
50 50 59
32*** 40*** 42***
50*** 66*** 63***
50*** 72*** 64***
a Number of individuals. NS: not significant (P > 0.05) between the control and UV treatments within the same column by Fisher’s exact test, DD: continuous darkness.
Number of individuals. P < 0.001 between the control and UV treatments within the same column by Fisher’s exact test, DD: continuous darkness.
significantly higher with UV-C and UV-B irradiances, as high as 0.6 W m2 (P < 0.01) and 2.4 W m2 (P < 0.001), respectively, compared to those under DD. In diapausing females, differences between mortality in all the UV treatments and those under DD were not significant (P > 0.05), even on day 3 (Table 3).
3.3. Oviposition
3.2. Escape In non-diapausing females, the incidences of escape were significantly higher with UV-C irradiance, as high as 0.6 W m2 on day 2 (P < 0.001), and 0.2 and 0.6 W m2 on day 3 (P < 0.05), compared to those under DD (Table 4). When the UV-B irradiance was as high as 2.4 W m2, the escape incidences were significantly higher than those under DD on days 2 and 3 (P < 0.01). When the UV-A irradiance was as high as 2.4 W m2, the escape incidence was significantly higher than that under DD, even on day 1 (P < 0.01). A significant difference was also found between the escape incidence with UV-A irradiance as low as 0.2 W m2 and that under DD on days 2 (P < 0.01) and 3 (P < 0.05). In diapausing females, significant differences were found between the escape incidence in all the UV treatments and those under DD, even on day 1 (P < 0.001), and almost half of the diapausing females exposed to UV radiation escaped from the leaf disks on day 3 (Table 5).
a
***
Oviposition on day 1 was significantly higher under UV-C (P < 0.05, Dunnett’s test) and UV-B (P < 0.01) irradiance of <0.2 W m2 than under DD (Table 6). Although low doses of UVC and UV-B might stimulate oviposition, the reason for this remains unknown. In contrast, even when the UV-C and UV-B irradiances were as low as 0.06 W m2 and 0.2 W m2, respectively, the oviposition incidences were significantly lower that those under DD on days 2 (P < 0.05) and 3 (P < 0.001). Interestingly, such inhibitory effects were not observed in the case of UV-A, and even when the irradiance was as high as 2.4 W m2, oviposition was not significantly different from that under DD on day 3. 3.4. Dose–response curves A significant positive correlation was detected between the total UV dose for 3 days and mortality plus escape incidences in non-diapausing females in the UV-C (r2 = 0.99, P = 0.00050, correlation analysis) and UV-B (r2 = 0.99, P = 0.041) treatments (Fig. 2). The median effective doses for 50% mortality plus escape
Table 4 Daily changes in escape percentage in non-diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Irradiance [W m2]
Na
Cumulative escape [%] Day 1
Day 2
Day 3
Control (DD)
–
98
0
0
3
UV-C
0.06 0.2 0.6
60 65 65
0NS 2NS 3NS
2NS 2NS 15***
3NS 12* 49***
UV-B
0.2 0.6 2.4
60 59 60
0NS 0NS 5NS
2NS 5NS 10**
2NS 5NS 20**
UV-A
0.2 0.6 2.4
60 60 60
2NS 5NS 10**
10** 7* 17***
12* 10NS 17**
a
Number of individuals. P < 0.05. ** P < 0.01. *** P < 0.001. NS: not significant (P > 0.05) between the control and UV treatments within the same column by Fisher’s exact test, DD: continuous darkness. *
Fig. 2. Dose–response relationship between the total UV dose and mortality plus escape percentage in non-diapausing Tetranychus urticae adult females.
652
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Table 6 Daily changes in the number of eggs laid by non-diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Irradiance [W m2]
Number of eggs laid per day (number of surviving females) Day 1
Day 2 a
Day 3
Control (DD)
–
1.4 0.1
(97)
8.3 0.2
(96)
9.4 0.3
(91)
UV-C
0.06 0.2 0.6
2.0 0.1** 1.9 0.2* 1.8 0.1NS
(60) (64) (63)
6.0 0.3*** 3.4 0.3*** 1.9 0.2***
(59) (62) (51)
5.7 0.4*** 2.2 0.4*** 0.6 0.2***
(54) (53) (22)
UV-B
0.2 0.6 2.4
2.0 0.2** 1.7 0.1NS 1.8 0.2NS
(60) (58) (57)
7.2 0.2* 5.2 0.3*** 3.3 0.2***
(58) (55) (52)
6.9 0.3*** 3.6 0.3*** 1.6 0.3***
(57) (55) (29)
UV-A
0.2 0.6 2.4
1.5 0.1NS 1.6 0.1NS 1.8 0.2NS
(59) (57) (54)
8.2 0.3NS 8.8 0.3NS 8.3 0.3NS
(54) (55) (50)
9.1 0.3NS 9.6 0.3NS 9.2 0.4NS
(53) (51) (50)
a
Mean SE. P < 0.05. P < 0.01. *** P < 0.001. NS: not significant (P > 0.05) between the control and UV treatments within the same column by Dunnett’s test, DD: continuous darkness. *
**
incidence (ED50) in non-diapausing females were 21 and 104 kJ m2 for the UV-C and UV-B treatments, respectively. A significant negative correlation was detected between the total UV dose for 3 days and oviposition rates in the UV-C (r2 = 0.99, P = 0.0021) and UV-B (r2 = 0.97, P = 0.015) treatments (Fig. 3). The ED50 values for 50% suppression of the oviposition incidence under DD (9.6 eggs for 3 days) were 6.2 and 41 kJ m2 for the UV-C and UV-B treatments, respectively. 4. Discussion UV-B directly damages DNA and is a strong oxidative stressor because it induces the homolysis of hydrogen peroxide (H2O2) into hydroxyl radical (OH), which seems to be the most damaging of the free radicals (Tan et al., 2007). UV-B-induced damage can be expected to be lethal to small organisms such as mites. In our experiments with the mite T. urticae, we observed that UV-C (250 nm) and UV-B (300 nm) in certain doses promoted mortality (Table 2) and escape (Table 4) and inhibited oviposition (Table 6) in non-diapausing females. Barcelo (1981) also showed that both UVC (254 nm) and UV-B (315 nm) inhibited oviposition in nondiapausing females. In the present study, the above-mentioned effects of UV-C were stronger than those of UV-B; the ED50 values
Fig. 3. Dose–response relationship between the total UV dose and number of eggs laid by non-diapausing Tetranychus urticae adult females. The broken line indicates 50% oviposition incidence under continuous darkness (9.6 eggs for 3 days).
for 50% mortality plus escape incidence and 50% suppression of the oviposition rate under DD were 21 and 6.2 kJ m2, respectively (UV-C), and 104 and 41 kJ m2, respectively (UV-B) (Figs. 2 and 3). These values for the UV-B treatment were actually observed in the field for 2–5 days during summer (Fig. 4). Therefore, UV-Bcontaining solar radiation is probably critical for survival and oviposition in non-diapausing females. T. urticae inhabits the undersides of leaves with dense vegetation in summer. Most UV radiation is absorbed and reflected by leaves (Suzuki and Takeda, in press). Therefore, the underside of leaves is likely to be as a suitable environment for T. urticae to avoid the deleterious effects of UV radiation, particularly UV-B (UV-C is completely absorbed by the ozone layer). Thus, non-diapausing females can safely go through their reproductive cycle on the underside of leaves. Interestingly, the predatory mite, Typhlodromalus aripo hides in the apex during the day and emerges at night to forage for the herbivorous mite, Mononychellus tanajoa found on young cassava leaves (Onzo et al., 2003, 2009). According to Onzo et al. (2009), hiding in the apex during the day protects T. aripo against harsh environmental conditions, particularly UV-B. In autumn, leaves start turning yellow and red as winter approaches and finally fall. During this phenological event, the UVB level in the plant canopy would increase dramatically while adult females of T. urticae enter diapause as their body color changes
Fig. 4. Seasonal changes in the daily-integrated UV-B radiation at Tsukuba, Japan (368030 N). Data are expressed as mean SD values from 1997 to 2007, and they were obtained from the Japan Meteorological Agency.
T. Suzuki et al. / Journal of Insect Physiology 55 (2009) 649–654
from yellow-green to orange. In diapausing females, UV-C and UVB irradiation did not result in an increase in mortality (Table 3), while UV-C, UV-B, and also UV-A irradiation were observed to greatly increase the escape incidence (Table 5). It is known that diapausing females show negative phototaxis (Hussey and Parr, 1963; Parr and Hussey, 1966) and positive geotaxis (Foott, 1965), and that they migrate from the host plants to overwinter (Veerman, 1985). Since low escape incidences (less than 10%) were observed in diapausing females under DD (Table 5), the high escape incidence under UV radiation would be mainly due to negative phototaxis, not positive geotaxis. In greenhouses, diapausing females overwinter in cracks and crevices in house structures, supporting stakes, hollow stems and straws on beds, irrigation equipment, door locks, and pipe fittings (Hussey, 1972; French and Ludlam, 1973; Veerman, 1985). In open fields, diapausing females have been found in clods of soil in apple orchards (Weldon, 1910), hop gardens (Massee, 1942), and blackcurrant plantations (Collingwood, 1955), and in clay soils (Helle, 1962). Diapausing females have also been found in the cracks of trees and under the bark of poles (Massee, 1942; Helle, 1962), on dried leaves (Massee, 1942) and straw (Collingwood, 1955), in the ground litter and cover (Kim and Lee, 2003), and in hollow withered flower stems (Helle, 1962). Moreover, overwintering females have been found under the bark in woody host plants (Helle, 1962; Uchida, 1980; Kim and Lee, 2003). The abovementioned habitats where diapausing females overwinter seem to offer a refuge from UV radiation. The best way to avoid the deleterious effects of UV radiation is by migrating to UV-free environments. Therefore, the high escape incidences observed in diapausing females under UV radiation (Table 5) may be an adaptation for surviving several months during winter in the absence of leaves as UV-cut filters. However, in the present study, we were unable to explain why the females could respond to UV-C, which does not reach the earth’s surface. Nevertheless, it is possible that UV-C-induced damage might affect their behavior. It is known that the orange color of diapausing females is attributable to the accumulation of carotenoids (Veerman, 1974), which act as a scavenger for ROS (Edge et al., 1997). Therefore, the low mortality observed in diapausing females under UV-C and UVB may be a result of carotenoid accumulation or merely an increase in the escape incidence. However, it is still unclear whether the diapausing females who escaped were resistant to UV-C and UV-B damage. Interestingly, Panonychus mites, which exhibit red body color, are often found not only on the underside but also on the upper side of leaves (Gutierrez and Helle, 1985). Moreover, the Texas citrus mite, Eutetranychus banksi exhibits tan to brownishgreen body color, and its populations are much heavier on the upper side of leaves, especially on the sunny sides of trees (Muma et al., 1953). The upper sides of leaves likely present a harsh UV environment for mites. Therefore, although the pigments present in Panonychus mites and E. banksi are still unknown, there might be a causal relationship between body color and UV tolerance. Recently, Suzuki et al. (2008b) reported that in non-diapausing females of T. urticae, melatonin (N-acetyl-5-methoxytryptamine) and its synthetic enzyme arylalkylamine N-acetyltransferase (EC 2.3.1.87) were activated by exposure to high dose UV-B. Moreover, it is known that melatonin functions as an ROS scavenger (Reiter et al., 2000). Therefore, not only carotenoid accumulation but also a melatonin response may be related to the differences in UV tolerance between non-diapausing and diapausing females. Thus, melatonin analysis of diapausing females is also required. In conclusion, our findings suggest that UV-B irradiation is an effective non-chemical measure for the reduction of the T. urticae populations, and that the selection of habitat and change in body color are strategies adopted by T. urticae to reduce the deleterious effects of UV-B.
653
Acknowledgements This work was conducted at the National Institute for Basic Biology Cooperative Researches on the Okazaki Large Spectrograph and supported by a Grant-in-Aid for JSPS Fellows (20-777). References Barcelo, J.A., 1981. Photoeffects of visible and ultraviolet radiation on the twospotted spider mite Tetranychus urticae. Photochemistry and Photobiology 33, 703–706. Barcelo, J.A., Calkins, J., 1980. The kinetics of avoidance of simulated solar UV radiation by two arthropods. Biophysical Journal 32, 921–929. Collingwood, C.A., 1955. The glasshouse red spider mite on blackcurrants. Annals of Applied Biology 43, 144–147. Edge, R., McGarvey, D.J., Truscott, T.G., 1997. The carotenoids as anti-oxidants—a review. Journal of Photochemistry and Photobiology B: Biology 41, 189–200. French, N., Ludlam, F.A.B., 1973. Observation on winter survival and diapausing behavior of red spider mite (Tetranychus urticae) on glasshouse roses. Plant Pathology 22, 16–21. Foott, W.H., 1965. 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kanzawa spider mite, Tetranychus kanzawai Kishida on pear tree in Tottori district (Acari: Tetranychidae). Japanese Journal of Applied Entomology and Zoology 24, 175–183 (in Japanese with English abstract). van de Vrie, M., McMurtry, J.A., Huffaker, C.B., 1972. Ecology of Tetranychid mites and their natural enemies: a review III. Biology, ecology, and pest status, and host–plant relations of tetranychids. Hilgardia 41, 343–432. Veerman, A., 1974. Carotenoid metabolism in Tetranychus urticae Koch (Acari: Tetranychidae). Comparative biochemistry and physiology B. Comparative Biochemistry 47, 101–116.
Veerman, A., 1985. Diapause. In: Helle, W., Sabelis, M.W. (Eds.), Spider Mites: Their Biology, Natural Enemies and Control, vol. 1A. Elsevier, Amsterdam, pp. 279– 316. Watanabe, M., Furuya, M., Miyoshi, Y., Inoue, Y., Iwahashi, I., Matsumoto, K., 1982. Design and performance of the Okazaki large spectrograph for photobiological research. Photochemistry and Photobiology 36, 491–498. Weldon, G.P., 1910. Life history notes and control of the common orchard mites Tetranychus bimaculatus and Bryobia pratensis. Journal of Economic Entomology 3, 430–434.
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
UV tolerance in the two-spotted spider mite, Tetranychus urticae Takeshi Suzuki a,*, Masakatsu Watanabe b,c, Makio Takeda a a
Graduate School of Agricultural Science, Kobe University, Rokko-dai, Nada, Kobe 657-8501, Japan National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 444-8585, Japan c Department of Photoscience, School of Advanced Sciences, Graduate University for Advanced Studies, Shonan Village, Hayama, Kanagawa 240-0193, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2009 Received in revised form 14 April 2009 Accepted 14 April 2009
The two-spotted spider mite, Tetranychus urticae was exposed to UV-C (250 nm), UV-B (300 nm), and UV-A (350 nm). In non-diapausing females, the median effective doses for 50% mortality plus escape incidence (ED50) were 21 (UV-C) and 104 kJ m2 (UV-B); those for 50% oviposition rate in continuous darkness-treated mites were 6.2 (UV-C) and 41 kJ m2 (UV-B). No significant effects of UV-A on mortality and oviposition rate were observed. The ED50 values for UV-B were similar to the natural UV-B observed for 2–5 days in summer when T. urticae inhabits the undersides of leaves. Therefore, T. urticae possibly uses leaves as a filter to avoid the deleterious effects of UV-B. In diapausing females, low mortality was observed even at high doses of UV radiation, but more than half escaped even at low doses. The orange body color of diapausing females results from accumulation of carotenoids, a scavenger for UV-induced reactive oxygen species; this may explain the low mortality of diapausing females. Diapausing females may overcome the deleterious effects of UV-B during winter in the absence of leaves by emigrating to UV-free environments and by accumulating carotenoids. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: UV irradiation Diapause Pest control Phototaxis Tetranychus urticae
1. Introduction Ultraviolet (UV) radiation directly damages DNA and is absorbed by some coenzymes and pigments in vivo; UV absorption raises these compounds to an excited state, and the excitation energy is finally transferred to H2O molecules, yielding reactive oxygen species (ROS). It has been suggested that UV-induced ROS damages important intra- and extracellular components, such as lipids, lipid membranes, nucleic acids, and proteins (Jurkiewicz and Buettner, 1994; Shindo et al., 1994). Such UV/ROS-induced damage would be lethal to mites because of their small size; i.e., their body surface area per weight is large. Therefore, artificial UV irradiation could prove to be a promising non-chemical measure for reducing populations of the two-spotted spider mite (Tetranychus urticae), which is distributed worldwide and causes serious damage to a wide variety of crops (van de Vrie et al., 1972). There is abundant information on the response to light, particularly photoperiodism, in T. urticae. Only adult females enter diapause in order to survive during winter by sensing longnight conditions, and diapausing females, which exhibit a orange body color in this period, do not feed or oviposit (Veerman, 1985). Response to UV radiation in T. urticae has been investigated with regard to behavior (McEnroe and Dronka, 1966; Naegele et al.,
* Corresponding author. Tel.: +81 78 803 5870; fax: +81 78 803 5870. E-mail address: [email protected] (T. Suzuki). 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.04.005
1966; Barcelo and Calkins, 1980; Barcelo, 1981), tolerance (Barcelo, 1981), and light-dependent enzyme activity (Suzuki et al., 2008b, 2009). However, these experiments were conducted with non-diapausing females, and data on the response of diapausing females to UV radiation is limited. The present study investigates differences in tolerance to UV radiation between non-diapausing and diapausing adult females and discusses phenological adaptations to UV radiation in T. urticae. 2. Materials and methods 2.1. Laboratory culture of T. urticae The founder population of T. urticae was collected from an apple (Malus pumila Mill. cv. Fuji) orchard located in Akita, Japan (398150 N), in 2001. The offspring population was maintained in the laboratory on kidney bean leaves (Phaseolus vulgaris L.) under longday (LD 16:8) conditions provided by white fluorescent lamps at 25 8C. The adult females that emerged from the teleiochrysalis stage within 3 days were used in the following UV tolerance experiment. To obtain diapausing females, adult females were placed on a fresh kidney bean leaf that was positioned on water-soaked cotton in a plastic Petri dish (diameter, 9 cm; depth, 2 cm); they were kept there 12 h under continuous darkness (DD) at 25 8C for oviposition and were removed after eggs were laid. The eggs were maintained for 5 days under the same environmental conditions; then, the
T. Suzuki et al. / Journal of Insect Physiology 55 (2009) 649–654
650
larvae that hatched were exposed to short-day (LD 8:16) conditions provided by white light-emitting diodes (LEDs) (NNN28300; Panasonic Electric Works Co. Ltd., Osaka, Japan) at 18 8C. Diapause induction was determined 7–10 days after adult emergence, and the adult females with uniform orange coloration were used in the following UV tolerance experiment. The nondiapausing and diapausing females were then placed on fresh leaf disks (diameter, 1 cm; 1 individual per disk) kept over watersoaked cotton in the plastic Petri dish. 2.2. UV irradiation The effects of UV radiation on the mortality, escape, and oviposition rates in T. urticae were determined by using the Okazaki Large Spectrograph (Watanabe et al., 1982) at the National Institute for Basic Biology, Okazaki, Japan. Monochromatic light was provided by a large spectrograph equipped with a 30 kW xenon arc lamp (Ushio Electric Co., Tokyo, Japan). The light beam was first reflected by a plane mirror and then by a condensing mirror. After reflection by a diffraction grating, the light beam passed into the irradiation boxes. In each Petri dish placed in the boxes with radiation wavelengths of 250, 300, and 350 nm, T. urticae samples were placed beneath a mirror that reflected the monochromatic light beam directly onto them. Table 1 lists the types of treatment, peak wavelength, irradiance, and dosages. Fig. 1 shows the spectral distributions of the treatments when irradiance ranging from 200 to 800 nm was set at 100 W m2. The irradiance values (0.06–0.6 W m2 for UV-C and 0.2– 2.4 W m2 for UV-B and UV-A) at specific wavelengths were
adjusted with UV filters (Fujitok Co., Tokyo, Japan) and measured with a radiation sensor (QTM-101; Monotech Inc., Saitama, Japan). Samples were treated under a light–dark cycle (LD 4:20) with monochromatic light of different irradiances for 3 days. 2.3. Observations After daily irradiation, the numbers of non-diapausing and diapausing females that were alive, dead, or had escaped, and the number of eggs laid by non-diapausing females were observed with a stereomicroscope (SZ-PT; Olympus Co., Tokyo, Japan). Viability or mortality was judged with the help of a brush; individuals that showed no reaction on contact with the brush were regarded as dead. Individuals that had fallen off the leaf disk onto the water-soaked cotton were considered to have escaped. The observation was conducted under the red light (>600 nm) that T. urticae could not sense (Suzuki et al., 2008a); thus, the undesirable effects of light were prevented. 2.4. Statistical analysis Statistical significances of differences between mortality and escape incidence under DD (control) and those exposed to different irradiances at a specific wavelength were determined by Fisher’s exact test. The statistical significance of the differences between the oviposition rate under DD and those exposed to different irradiances at a specific wavelength was determined by one-way ANOVA and then Dunnett’s test. A correlation analysis was conducted to determine the UV dose response in terms of mortality, escape, and oviposition. The calculations were performed using SigmaPlot 2001 (SPSS Inc., Chicago, IL) and SPSS 11.5J software (SPSS Japan Inc., Tokyo, Japan). 3. Results 3.1. Mortality In non-diapausing females, differences between mortality in all UV treatments and those in the control conditions (DD) were not significant on days 1 and 2 (P > 0.05, Fisher’s exact test); the mortality was nearly 0% (Table 2). Even on day 3, mortality in the UV-C, UV-B, and UV-A treatments with irradiances below 0.2, 0.6, and 2.4 W m2, respectively, was not significantly different from those under DD (P > 0.05). However, mortality on day 3 was
Fig. 1. Spectral distributions of the irradiation treatments when the irradiance was set at 100 W m2.
Table 2 Daily changes in mortality in non-diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Irradiance [W m2]
Na
Cumulative mortality [%] Day 1
Table 1 Type of UV radiation, peak wavelength, irradiance, and daily-integrated dose.
Day 3
Control (DD)
–
98
1
2
4
UV-C
0.06 0.2 0.6
60 65 65
0NS 0NS 0NS
0NS 3NS 3NS
7NS 6NS 18**
0.9 2.9 8.6
UV-B
0.2 0.6 2.4
60 59 60
0NS 2NS 0NS
2NS 2NS 3NS
3NS 2NS 32***
UV-A
0.2 0.6 2.4
60 60 60
0NS 0NS 0NS
0NS 2NS 0NS
0NS 5NS 0NS
Type of UV radiation
Peak wavelength [nm]
Irradiance [W m2]
UV-C
250
0.06 0.2 0.6
UV-B
300
0.2 0.6 2.4
2.9 8.6 34.6
UV-A
350
0.2 0.6 2.4
2.9 8.6 34.6
The exposure time was 4 h per day.
Day 2
Daily-integrated dose [kJ m2 day1]
a
Number of individuals. P < 0.01. P < 0.001, NS: not significant (P > 0.05) between the control and UV treatments within the same column by Fisher’s exact test, DD: continuous darkness. **
***
T. Suzuki et al. / Journal of Insect Physiology 55 (2009) 649–654 Table 3 Daily changes in mortality in diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Irradiance [W m2]
Na
Day 1
Day 2
Table 5 Daily changes in escape percentage in diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Cumulative mortality [%]
651
Day 3
Irradiance [W m2]
Na
Cumulative escape [%] Day 1
Day 2
Day 3
Control (DD)
–
72
0
1
1
Control (DD)
–
72
3
8
10
UV-C
0.06 0.2 0.6
52 57 65
6NS 2NS 0NS
10NS 4NS 0NS
10NS 5NS 6NS
UV-C
0.06 0.2 0.6
52 57 65
19*** 21*** 14***
35*** 35*** 42***
38** 40*** 51***
UV-B
0.2 0.6 2.4
50 52 60
0NS 0NS 0NS
4NS 2NS 3NS
6NS 6NS 5NS
UV-B
0.2 0.6 2.4
50 52 60
38*** 27*** 28***
68*** 42*** 52***
68*** 44*** 63***
UV-A
0.2 0.6 2.4
50 50 59
0NS 2NS 0NS
2NS 8NS 3NS
2NS 8NS 8NS
UV-A
0.2 0.6 2.4
50 50 59
32*** 40*** 42***
50*** 66*** 63***
50*** 72*** 64***
a Number of individuals. NS: not significant (P > 0.05) between the control and UV treatments within the same column by Fisher’s exact test, DD: continuous darkness.
Number of individuals. P < 0.001 between the control and UV treatments within the same column by Fisher’s exact test, DD: continuous darkness.
significantly higher with UV-C and UV-B irradiances, as high as 0.6 W m2 (P < 0.01) and 2.4 W m2 (P < 0.001), respectively, compared to those under DD. In diapausing females, differences between mortality in all the UV treatments and those under DD were not significant (P > 0.05), even on day 3 (Table 3).
3.3. Oviposition
3.2. Escape In non-diapausing females, the incidences of escape were significantly higher with UV-C irradiance, as high as 0.6 W m2 on day 2 (P < 0.001), and 0.2 and 0.6 W m2 on day 3 (P < 0.05), compared to those under DD (Table 4). When the UV-B irradiance was as high as 2.4 W m2, the escape incidences were significantly higher than those under DD on days 2 and 3 (P < 0.01). When the UV-A irradiance was as high as 2.4 W m2, the escape incidence was significantly higher than that under DD, even on day 1 (P < 0.01). A significant difference was also found between the escape incidence with UV-A irradiance as low as 0.2 W m2 and that under DD on days 2 (P < 0.01) and 3 (P < 0.05). In diapausing females, significant differences were found between the escape incidence in all the UV treatments and those under DD, even on day 1 (P < 0.001), and almost half of the diapausing females exposed to UV radiation escaped from the leaf disks on day 3 (Table 5).
a
***
Oviposition on day 1 was significantly higher under UV-C (P < 0.05, Dunnett’s test) and UV-B (P < 0.01) irradiance of <0.2 W m2 than under DD (Table 6). Although low doses of UVC and UV-B might stimulate oviposition, the reason for this remains unknown. In contrast, even when the UV-C and UV-B irradiances were as low as 0.06 W m2 and 0.2 W m2, respectively, the oviposition incidences were significantly lower that those under DD on days 2 (P < 0.05) and 3 (P < 0.001). Interestingly, such inhibitory effects were not observed in the case of UV-A, and even when the irradiance was as high as 2.4 W m2, oviposition was not significantly different from that under DD on day 3. 3.4. Dose–response curves A significant positive correlation was detected between the total UV dose for 3 days and mortality plus escape incidences in non-diapausing females in the UV-C (r2 = 0.99, P = 0.00050, correlation analysis) and UV-B (r2 = 0.99, P = 0.041) treatments (Fig. 2). The median effective doses for 50% mortality plus escape
Table 4 Daily changes in escape percentage in non-diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Irradiance [W m2]
Na
Cumulative escape [%] Day 1
Day 2
Day 3
Control (DD)
–
98
0
0
3
UV-C
0.06 0.2 0.6
60 65 65
0NS 2NS 3NS
2NS 2NS 15***
3NS 12* 49***
UV-B
0.2 0.6 2.4
60 59 60
0NS 0NS 5NS
2NS 5NS 10**
2NS 5NS 20**
UV-A
0.2 0.6 2.4
60 60 60
2NS 5NS 10**
10** 7* 17***
12* 10NS 17**
a
Number of individuals. P < 0.05. ** P < 0.01. *** P < 0.001. NS: not significant (P > 0.05) between the control and UV treatments within the same column by Fisher’s exact test, DD: continuous darkness. *
Fig. 2. Dose–response relationship between the total UV dose and mortality plus escape percentage in non-diapausing Tetranychus urticae adult females.
652
T. Suzuki et al. / Journal of Insect Physiology 55 (2009) 649–654
Table 6 Daily changes in the number of eggs laid by non-diapausing Tetranychus urticae adult females under continuous darkness and UV radiation. Treatment
Irradiance [W m2]
Number of eggs laid per day (number of surviving females) Day 1
Day 2 a
Day 3
Control (DD)
–
1.4 0.1
(97)
8.3 0.2
(96)
9.4 0.3
(91)
UV-C
0.06 0.2 0.6
2.0 0.1** 1.9 0.2* 1.8 0.1NS
(60) (64) (63)
6.0 0.3*** 3.4 0.3*** 1.9 0.2***
(59) (62) (51)
5.7 0.4*** 2.2 0.4*** 0.6 0.2***
(54) (53) (22)
UV-B
0.2 0.6 2.4
2.0 0.2** 1.7 0.1NS 1.8 0.2NS
(60) (58) (57)
7.2 0.2* 5.2 0.3*** 3.3 0.2***
(58) (55) (52)
6.9 0.3*** 3.6 0.3*** 1.6 0.3***
(57) (55) (29)
UV-A
0.2 0.6 2.4
1.5 0.1NS 1.6 0.1NS 1.8 0.2NS
(59) (57) (54)
8.2 0.3NS 8.8 0.3NS 8.3 0.3NS
(54) (55) (50)
9.1 0.3NS 9.6 0.3NS 9.2 0.4NS
(53) (51) (50)
a
Mean SE. P < 0.05. P < 0.01. *** P < 0.001. NS: not significant (P > 0.05) between the control and UV treatments within the same column by Dunnett’s test, DD: continuous darkness. *
**
incidence (ED50) in non-diapausing females were 21 and 104 kJ m2 for the UV-C and UV-B treatments, respectively. A significant negative correlation was detected between the total UV dose for 3 days and oviposition rates in the UV-C (r2 = 0.99, P = 0.0021) and UV-B (r2 = 0.97, P = 0.015) treatments (Fig. 3). The ED50 values for 50% suppression of the oviposition incidence under DD (9.6 eggs for 3 days) were 6.2 and 41 kJ m2 for the UV-C and UV-B treatments, respectively. 4. Discussion UV-B directly damages DNA and is a strong oxidative stressor because it induces the homolysis of hydrogen peroxide (H2O2) into hydroxyl radical (OH), which seems to be the most damaging of the free radicals (Tan et al., 2007). UV-B-induced damage can be expected to be lethal to small organisms such as mites. In our experiments with the mite T. urticae, we observed that UV-C (250 nm) and UV-B (300 nm) in certain doses promoted mortality (Table 2) and escape (Table 4) and inhibited oviposition (Table 6) in non-diapausing females. Barcelo (1981) also showed that both UVC (254 nm) and UV-B (315 nm) inhibited oviposition in nondiapausing females. In the present study, the above-mentioned effects of UV-C were stronger than those of UV-B; the ED50 values
Fig. 3. Dose–response relationship between the total UV dose and number of eggs laid by non-diapausing Tetranychus urticae adult females. The broken line indicates 50% oviposition incidence under continuous darkness (9.6 eggs for 3 days).
for 50% mortality plus escape incidence and 50% suppression of the oviposition rate under DD were 21 and 6.2 kJ m2, respectively (UV-C), and 104 and 41 kJ m2, respectively (UV-B) (Figs. 2 and 3). These values for the UV-B treatment were actually observed in the field for 2–5 days during summer (Fig. 4). Therefore, UV-Bcontaining solar radiation is probably critical for survival and oviposition in non-diapausing females. T. urticae inhabits the undersides of leaves with dense vegetation in summer. Most UV radiation is absorbed and reflected by leaves (Suzuki and Takeda, in press). Therefore, the underside of leaves is likely to be as a suitable environment for T. urticae to avoid the deleterious effects of UV radiation, particularly UV-B (UV-C is completely absorbed by the ozone layer). Thus, non-diapausing females can safely go through their reproductive cycle on the underside of leaves. Interestingly, the predatory mite, Typhlodromalus aripo hides in the apex during the day and emerges at night to forage for the herbivorous mite, Mononychellus tanajoa found on young cassava leaves (Onzo et al., 2003, 2009). According to Onzo et al. (2009), hiding in the apex during the day protects T. aripo against harsh environmental conditions, particularly UV-B. In autumn, leaves start turning yellow and red as winter approaches and finally fall. During this phenological event, the UVB level in the plant canopy would increase dramatically while adult females of T. urticae enter diapause as their body color changes
Fig. 4. Seasonal changes in the daily-integrated UV-B radiation at Tsukuba, Japan (368030 N). Data are expressed as mean SD values from 1997 to 2007, and they were obtained from the Japan Meteorological Agency.
T. Suzuki et al. / Journal of Insect Physiology 55 (2009) 649–654
from yellow-green to orange. In diapausing females, UV-C and UVB irradiation did not result in an increase in mortality (Table 3), while UV-C, UV-B, and also UV-A irradiation were observed to greatly increase the escape incidence (Table 5). It is known that diapausing females show negative phototaxis (Hussey and Parr, 1963; Parr and Hussey, 1966) and positive geotaxis (Foott, 1965), and that they migrate from the host plants to overwinter (Veerman, 1985). Since low escape incidences (less than 10%) were observed in diapausing females under DD (Table 5), the high escape incidence under UV radiation would be mainly due to negative phototaxis, not positive geotaxis. In greenhouses, diapausing females overwinter in cracks and crevices in house structures, supporting stakes, hollow stems and straws on beds, irrigation equipment, door locks, and pipe fittings (Hussey, 1972; French and Ludlam, 1973; Veerman, 1985). In open fields, diapausing females have been found in clods of soil in apple orchards (Weldon, 1910), hop gardens (Massee, 1942), and blackcurrant plantations (Collingwood, 1955), and in clay soils (Helle, 1962). Diapausing females have also been found in the cracks of trees and under the bark of poles (Massee, 1942; Helle, 1962), on dried leaves (Massee, 1942) and straw (Collingwood, 1955), in the ground litter and cover (Kim and Lee, 2003), and in hollow withered flower stems (Helle, 1962). Moreover, overwintering females have been found under the bark in woody host plants (Helle, 1962; Uchida, 1980; Kim and Lee, 2003). The abovementioned habitats where diapausing females overwinter seem to offer a refuge from UV radiation. The best way to avoid the deleterious effects of UV radiation is by migrating to UV-free environments. Therefore, the high escape incidences observed in diapausing females under UV radiation (Table 5) may be an adaptation for surviving several months during winter in the absence of leaves as UV-cut filters. However, in the present study, we were unable to explain why the females could respond to UV-C, which does not reach the earth’s surface. Nevertheless, it is possible that UV-C-induced damage might affect their behavior. It is known that the orange color of diapausing females is attributable to the accumulation of carotenoids (Veerman, 1974), which act as a scavenger for ROS (Edge et al., 1997). Therefore, the low mortality observed in diapausing females under UV-C and UVB may be a result of carotenoid accumulation or merely an increase in the escape incidence. However, it is still unclear whether the diapausing females who escaped were resistant to UV-C and UV-B damage. Interestingly, Panonychus mites, which exhibit red body color, are often found not only on the underside but also on the upper side of leaves (Gutierrez and Helle, 1985). Moreover, the Texas citrus mite, Eutetranychus banksi exhibits tan to brownishgreen body color, and its populations are much heavier on the upper side of leaves, especially on the sunny sides of trees (Muma et al., 1953). The upper sides of leaves likely present a harsh UV environment for mites. Therefore, although the pigments present in Panonychus mites and E. banksi are still unknown, there might be a causal relationship between body color and UV tolerance. Recently, Suzuki et al. (2008b) reported that in non-diapausing females of T. urticae, melatonin (N-acetyl-5-methoxytryptamine) and its synthetic enzyme arylalkylamine N-acetyltransferase (EC 2.3.1.87) were activated by exposure to high dose UV-B. Moreover, it is known that melatonin functions as an ROS scavenger (Reiter et al., 2000). Therefore, not only carotenoid accumulation but also a melatonin response may be related to the differences in UV tolerance between non-diapausing and diapausing females. Thus, melatonin analysis of diapausing females is also required. In conclusion, our findings suggest that UV-B irradiation is an effective non-chemical measure for the reduction of the T. urticae populations, and that the selection of habitat and change in body color are strategies adopted by T. urticae to reduce the deleterious effects of UV-B.
653
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