Effects of Low Temperature on Spider Mite Control by Intermittent Ultraviolet-B Irradiation for Practical Use in Greenhouse Strawberries

Effects of Low Temperature on Spider Mite Control by Intermittent Ultraviolet-B Irradiation for... Abstract The application of ultraviolet-B (UVB) radiation to control spider mites is challenging as a key technology for integrated pest management (IPM) in greenhouse strawberries in Japan. To address this, concurrent use of phytoseiid mites and reduced UVB irradiance is desirable to ensure control effects in areas shaded from UVB radiation and to minimize the sunscald in winter, respectively. We designed experiments reproducing the UVB dose on the lower leaf surfaces in strawberry and evaluated the effects of intermittent UVB irradiation at midnight for practical application in the greenhouse and low temperature on the survival of the spider mite Tetranychus urticae Koch (Acari: Tetranychidae) and damage to the phytoseiid mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae). The midnight intermittent UVB irradiation effectively suppressed egg hatching and development of larvae of T. urticae, and the control effect was reinforced at 20°C (no eggs hatched at 0.13 kJ m−2 d−1) rather than, at 25°C (70.8% eggs hatched). In contrast, the hatchability of N. californicus eggs was unaffected by intermittent UVB irradiation at 0.27 kJ m−2 d−1 at 25°C and 20°C. However, residual effects of UVB irradiation to N. californicus eggs on survival of hatched larvae were seen, so that reducing the UVB dose is also advantageous for this phytoseiid mite. N. californicus showed a photoreactivation capacity, whereas their UVB tolerance was improved by prey species, suggesting the possibility of the improvement of phytoseiid mites by diet. The reduction of UVB dose and concurrent use of phytoseiid mites increase reliability of the UVB method in IPM strategies in strawberry greenhouse. Deleterious effects of ultraviolet-B (UVB; 280–315 nm) radiation on spider mites have been reported over the last decade (Suzuki et al. 2009, Ohtsuka and Osakabe 2009, Sakai et al. 2012, Murata and Osakabe 2017). A UVB lamp was developed to control powdery mildew in greenhouses (Kanto et al. 2009, 2014; Kobayashi et al. 2013). Conspicuous control effects on spider mite populations by the combined use of UVB lamps and light reflection sheets were demonstrated in melons (Masui et al. 2013) and strawberries (Tanaka et al. 2016) in greenhouses. Spider mites and powdery mildew are economically important pest and disease, respectively, especially in strawberries, so that simultaneous control of these pest and disease is required. Moreover, the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae), a major mite pest in strawberry, has developed serious resistance to the most acaricides (Van Leeuwen et al. 2010, 2015). A control method which can simultaneously control spider mites and powdery mildew may be a good candidate for integrated pest management (IPM) in greenhouses, especially for strawberry production. Tanaka et al. (2016) used wide intervals of rows (0.4–0.5 m) and plants (0.3–0.35 m) to make it easier for UVB radiation to reach lower leaf surfaces where spider mites reside. In contrast, under economically favorable narrower row- (0.25 m) and plant-interval (0.23–0.25 m) conditions, leaves may become overgrown, increasing the region of leaves shaded from UVB radiation. Increasing UVB intensity may decrease the shaded area on overgrown leaves. However, excessive UVB irradiation may increase plant damage such as sunscald occurred in strawberry leaves in winter (Tanaka et al. 2016). In practical terms and for effective IPM, concurrent use of phytoseiid mites with UVB and reduction in UVB irradiance to minimize the sunscald in winter are desirable. Studies indicated high vulnerability to UVB radiation in phytoseiid mites (Onzo et al. 2010, Ghazy et al. 2016); their prompt avoidance from areas irradiated with UVB has also been suggested (Tachi and Osakabe 2012, 2014). In these studies, vulnerability was tested using a single acute irradiation of UVB. Unlike these, UVB irradiation was performed for 3 h at midnight in strawberry greenhouses (Tanaka et al. 2016). Spider mites are potentially reactivated by photo-enzymatic repair (PER) of DNA lesions, using energy from visible light (VIS) and ultraviolet-A (UVA; 315–400) in sunshine during the day under greenhouse conditions (Suzuki et al. 2014; Murata and Osakabe 2014, 2017). However, the effects of photoreactivation were reduced with increasing time lag between UVB irradiation and exposure to VIS in eggs, and no reactivation effects remained when the time lag was 4 h (Murata and Osakabe 2014). For that reason Tanaka et al. (2016) irradiated strawberries with UVB at midnight and ended 3–4 h before sunrise, whereas the 4-h time lag did not interfere with the photoreactivation of larvae (Murata and Osakabe 2014). Thus, this method may be effective suppressing egg hatching while less effective in interfering with juvenile development of spider mites. In phytoseiid mites, Koveos et al. (2017) tested the effects of photoreactivation under simultaneous UVB and white light radiation. Unlike T. urticae, the white light radiation did not increase egg hatchability of phytoseiid mites, indicating that the phytoseiid mites were more sensitive to UVB damage than spider mites and did not rely on a photoreactivation system (Koveos et al. 2017). Despite demand to reduce UVB irradiance in winter, the effects of low temperature on survival and DNA repair in mites exposed to UVB radiation are largely unknown. According to studies in other organisms, the effects of temperature on damage and repair are unlikely to be simple responses. Survival and photoreactivation rates of freshwater Daphnia species (Cladocera, Daphiidae) were higher at 10°C than at 20°C (Connelly et al. 2009). Similarly, the yield of DNA damage after exposure to UVB radiation at 12°C is lower than that at 24°C in a marine bacterium Shingopyxis alaskensis (Matallana-Surget et al. 2010). In contrast, Escherichia coli cells grown at 20°C are more sensitive to UVB radiation than those grown at 37°C (Mangoli et al. 2014). Nevertheless, there are reports of higher vulnerability to UVB radiation of Daphnia species at lower temperatures (Williamson et al. 2002, Macfadyen et al. 2004). In this study, we designed experiments reproduced the UVB dose on the lower leaf surfaces in strawberry greenhouse in a chamber and evaluated the effects of intermittent irradiation of UVB (3-h irradiation every night) and that of lower temperature (20°C) on the survival of T. urticae and the phytoseiid mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) in comparison with a higher temperature (25°C). We also tested the capacity for photoreactivation in phytoseiid mites. Materials and Methods Mites T. urticae and N. californicus were used for the evaluation of biological impact of UVB radiation, simulating the irradiation conditions in a greenhouse (Tanaka et al. 2016). A T. urticae population was obtained from several areas in Japan and was reared on kidney bean Phaseolus vulgaris L. leaves. The N. californicus strain had been originally collected from Japanese pear Pyrus pyrifolia (Burm.f.) Nakai trees in Matsukawa, Nagano Prefecture, Japan (35° 36ʹ N, 137° 55ʹ E) in September 2000 and was reared on the T. urticae population in the laboratory at 25°C (approximately 80% RH) under a photoperiod of 16:8 (L:D) h. In the experiments on photoreactivation, N. californicus was reared on both T. urticae and the citrus red mite Panonychus citri (McGregor) (Acari: Tetranychidae) for more than two generations prior to use. The P. citri population was collected from Satsuma mandarin Citrus unshiu Marc. (‘Tsunoka’) at the National Institute of Fruit Tree Science, Minamishimabara, Nagasaki, Japan (32° 36ʹ N, 130° 11ʹ E) on 1 June 2007 and reared on kidney bean leaves in the laboratory. All kidney bean leaves used for the rearing cultures and experiments were primary leaves. UVB and VIS Irradiation Conditions The UVB irradiance was measured using an irradiance meter (X11) equipped with a UV-3702–4 detector head (Gigahertz-Optik, Türkenfeld, Germany). The VIS irradiance in the photoreactivation experiment was measured using a photo-radiometer (HD2102.2) equipped with an irradiance measurement probe (LP 471 RAD; Delta OHM, Padova, Italy). Irradiation equipment was similar to that used by Murata and Osakabe (2014, 2017). A UVB lamp (6 W; Panasonic Electric Works, Osaka, Japan) was affixed from a shelf 67 cm overhead in a growth chamber at 25°C and 20°C. The growth chamber was illuminated with fluorescent lights (≈7,000 lx on the shelf) from 08:00 to 22:00 (simulated daytime; 14:10 [L:D] h) every day. The UVB lamp was turned on at 00:00 and turned off at 03:00 every day in the simulated nighttime. The UVB intensity of direct irradiation was 0.16 W m−2 on the shelf. The UVB lamp was covered with polyethylene sheets except during the photoreactivation experiment. Polyethylene sheets have no effects on the wavelength spectrum of UVB irradiation, and the intensity was adjusted to 0.012, 0.025, and 0.055 W m−2: UVB irradiances were 0.13, 0.27, and 0.59 kJ m−2 d−1, respectively. The UVB intensities of 0.025 and 0.055 W m−2 roughly corresponded with that on lower leaf surfaces of greenhouse strawberries (0.022–0.053 W m−2) in Tanaka et al. (2016). The daily cumulative UVB irradiances were equivalent to or below the LD50 value for T. urticae eggs (0.58 kJ m−2) reported by Murata and Osakabe (2013). A single acute UVB irradiation was performed with direct irradiation from the UVB lamp in the photoreactivation experiments. During photoreactivation experiments, the fluorescent lights were turned off; and, two halogen lamps (130 W; JDR110V-85WHM/K7-H; Ushio Lighting, Tokyo, Japan; set at an interval of 22.5 cm) affixed from a shelf 67 cm overhead in the dark growth chamber were used as VIS sources; the irradiance value was 67.7 W m−2 on the shelf. Effects of Daily UVB Irradiation and Temperature on T. urticae Egg Hatchability We prepared two Petri dishes (9 cm in diameter), one for UVB treatment and the other for non-irradiated control. Four pieces of squared kidney bean leaves (2 × 2 cm) were placed on water-soaked cotton in a Petri dish. Five adult T. urticae females were introduced to each kidney bean leaf. One Petri dish was placed in a plastic basket (20 × 27 × 8.5 cm) in which the top was covered with UV-opaque film (HB3 polyester film; DuPont Teijin Films, Chester, VA) (UV−) and the other Petri dish was placed in the same plastic basket covered with UVB-transparent film (polyvinylidene chloride film; Saran Wrap; Asahi Kasei Home Products Co., Tokyo, Japan) (UV+) and allowed to lay eggs in the growth chamber. The UV-opaque film filtered out more than 90% of UV at wavelengths less than 380 nm (Sakai and Osakabe 2010; Supplementary Fig. 1), whereas the UVB-transparent film transmitted more than 90% of UVB and VIS (Supplementary Fig. 1). Humidity was not adjusted and fluctuated between 45 and 65% RH in the plastic basket. After 24 h the adult females were removed and the numbers of eggs on the leaves were arranged to 20 (80 eggs per Petri dish; day 0). The eggs were kept in the same growth chamber and the number of hatched larvae and unhatched eggs were counted 6 d later (day 6). Experiments were performed for UVB irradiance of 0.13 and 0.27 kJ m−2 d−1 at 25°C and 0.13 kJ m−2 d−1 at 20°C. All experiments were replicated three times. Data sets were pooled over three replications. Variation of hatchability among treatments (0, 0.13, and 0.27 kJ m−2 d−1) at 25°C was analyzed with Fisher’s exact test using the ‘pairwise.fisher.test’ module of the ‘fmsb’ package and the ‘fisher.test’ module, in the ‘R’ software (ver. 3.3.3; R Core Team 2017). Survival of Larvae Two Petri dishes were prepared as described for T. urticae egg hatchability experiments, but the number of leaves per Petri dish was two at 20°C. Twenty T. urticae larvae that were within 3 h after hatching were introduced to each leaf (80 and 40 larvae per Petri dish for the experiments at 25°C and 20°C, respectively). The larvae were prepared by a method to synchronize egg hatching, as described in Ubara and Osakabe (2015), without UVB radiation. The Petri dishes were set in the plastic basket for UV− and UV+ and reared in the growth chamber until adulthood, death, or escape. Escaped individuals were omitted from the following data analysis. Experiments were performed for UVB irradiances of 0.27 and 0.55 kJ m−2 d−1 at 25°C and 0.27 kJ m−2 d−1 at 20°C. In the experiments with 0.27 kJ m−2 d−1 UVB at 25°C, we checked egg production by adult females randomly chosen from survivors (15 ♀♀ in the first replication and 30 ♀♀ in the second and third replications) individually reared on kidney bean leaves for 5 d after the first oviposition. All experiments were replicated three times. We pooled the data sets of the survival of larvae to adults and developed adult females over the replications. The difference of the survival between treatments with and without UVB irradiation was analyzed by Fisher’s exact test using the ‘fisher.test’ module in the R software. The effects of UVB radiation on egg production were evaluated by nested analysis of variance (nested ANOVA) after Bartlett’s test using the ‘aov’ and ‘bartlett.test’ modules in the R software. Effects of Daily UVB Irradiation and Temperature on Egg Hatchability of N. californicus We prepared two and three Petri dishes for the experiments at 25°C and 20°C, respectively. Each Petri dish contained a squared kidney bean leaf (4 × 4 cm), placed on water-soaked cotton. Ten adult female T. urticae were introduced to each leaf. The Petri dishes were set in the plastic baskets for UV− (two of three dishes were assigned at 20°C) and UV+ and placed in the growth chamber where irradiated with UVB at 0.025 W m−2 for 3 h (0.27 kJ m−2 d−1) every night. After 24 h, when T. urticae females had laid eggs, 10 adult females of N. californicus were introduced to each leaf and reared together with T. urticae females and their eggs for 24 h in the same chamber. Then, females of T. urticae and N. californicus were removed. Eggs of N. californicus laid on the leaf were continuously reared in the same chamber (day 0), and their hatchability was determined 3 d later (day 3) and 5 d later (day 5) in the experiments at 25°C and 20°C, respectively. All experiments were replicated three times. The numbers of eggs used for experiments were 65 (20–25 eggs per replication) and 72 (22–25) at 25°C and 67 (20–27) and 113 (33–40) at 20°C. The data sets over three replications were pooled, and differences in the hatchability between treatments with and without UVB irradiation were analyzed by Fisher’s exact test using the ‘fisher.test’ module in the R software. Effects of Daily UVB Irradiation at Egg Stage and After Hatching on Development of N. californicus Four Petri dishes were prepared in the same manner as that in the experiments on the hatchability of N. californicus eggs. Ten adult females of T. urticae were introduced to each leaf and two Petri dishes were set in the plastic basket for UV−, and the remaining two were in that for UV+ and placed in the growth chamber at 0.025 W m−2 for 3 h (0.27 kJ m−2 d−1) UVB irradiation at 25°C. After 24 h, 10 adult females of N. californicus were introduced to each leaf and allowed to lay eggs. The N. californicus females were removed from leaves. The Petri dishes containing N. californicus eggs and eggs and adult females of T. urticae on the leaves were kept continuously in baskets in the chamber. Three days later, unhatched N. californicus eggs were removed from leaves. Then, one of two Petri dishes in the UV− basket and one of two Petri dishes in the UV+ baskets were interchanged (day 0). The N. californicus larvae were reared until adult emergence. Developmental success was determined on day 4. By this method, we set four treatments: 1) irradiated with UVB from egg to adult (n = 17–21 [in each replication]); 2) irradiated from larva to adult but not irradiated during egg and the beginning of larval stage (n = 17–20); 3) irradiated during egg and the beginning of the larval stage, but not irradiated from larva to adult (n = 15–25); and 4) non-irradiated during egg to adult (n = 16–20; control). All experiments were replicated three times. Prior to data analysis, we performed an arc sine transformation and Bartlett’s test using the ‘bartlett.test’ module, then two-way ANOVA using the ‘aov’ module in the R software. Because unhatched eggs were excluded from the data analysis, we could evaluate the residual effects of UVB irradiation to eggs and direct effects of UVB irradiation on the juvenile stages of development. Photoreactivation in N. californicus Eggs Two Petri dishes were containing four pieces of squared kidney bean leaves (2 × 2 cm) on water-soaked cotton. Ten T. urticae adult females or 40 P. citri females were introduced to each leaf and placed in a laboratory at 25°C under a photoperiod of 16:8 (L:D) h. The spider mite females were removed after 24 h. The five adult females of N. californicus reared on T. urticae and P. citri were introduced to the leaves containing T. urticae eggs and P. citri eggs, respectively, and allowed to lay eggs for 24 h. Then, we removed phytoseiid mite females and immediately exposed their eggs to UVB radiation at 0.16 W m−2 for 30 min (0.29 kJ m−2) in the growth chamber. Immediately after the end of UVB irradiation, one Petri dish was put inside a cardboard box (dark box: 24.0 × 16.5 × 10.8 cm). The dark box containing a Petri dish (dark control) and the other Petri dish (photoreactivation treatment) were exposed to VIS (halogen lamp, 67.7 W m−2) for 90 min (365.6 kJ m−2). Then, the Petri dish for photoreactivation treatment was also put inside the cardboard box together with the dark control and placed in the laboratory (day 0). Eggs were observed every day, although the dark condition was interrupted briefly by the observation. Egg hatchability was determined 4 d later (day 4). Larvae that died soon after hatching were also counted on day 4, and used to calculate survival rates. All experiments were replicated twice. The numbers of N. californicus eggs reared on T. urticae were 52 and 50 for the dark control and 52 and 53 for photoreactivation treatment in the first and second replications, respectively; the numbers of N. californicus eggs reared on P. citri were 64 and 56 for dark control and 53 and 54 for the photoreactivation treatment. Effects of VIS irradiation after UVB irradiation on egg hatchability and survival of hatched larvae of N. californicus were evaluated by a generalized linear model (GLM; family = binomial) using the ‘glm’ module in the R software. Results Effects of Daily UVB Irradiation and Temperature on T. urticae Egg Hatchability The hatchability of eggs exposed to no UVB radiation (0 kJ m−2 d−1; UV−) was 91.3% and 92.1% at 25°C and 20°C, respectively (Fig. 1). In contrast, no egg hatched with UVB irradiation at 0.025 W m−2 (0.27 kJ m−2 d−1) at 25°C (Fisher’s exact test, P = 2 × 10−16; Fig. 1a). With UVB irradiation at 0.012 W m−2 (0.13 kJ m−2 d−1), egg hatchability decreased to 70.8% at 25°C (P = 1.2 × 10−8; Fig. 1a) and to zero at 20°C (P = 2.2 × 10−16; Fig. 1b). Fig. 1. View largeDownload slide Effects of UVB radiation on the hatchability of Tetranychus urticae eggs at (a) 25°C and (b) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Fig. 1. View largeDownload slide Effects of UVB radiation on the hatchability of Tetranychus urticae eggs at (a) 25°C and (b) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Survival of Larvae More than 94% of larvae exposed to no UVB radiation (0 kJ m−2 d−1; UV−) developed to adults at 25°C and 87.5% at 20°C (Fig. 2). Most larvae died with UVB irradiation at 0.055 W m−2 (0.59 kJ m−2 d−1) at 25°C (Fisher’s exact test, P = 2.2 × 10−16; Fig. 2b). With UVB irradiation at 0.025 W m−2 (0.27 kJ m−2 d−1), development was decreased slightly, to 84.6%, at 25°C (P = 1.93 × 10−6; Fig. 2a) and reduced significantly, to 36.7%, at 20°C (P = 1.87 × 10−11; Fig. 2c). Only one individual irradiated with UVB escaped from the leaf in the development experiment at 20°C; however, no individuals escaped in the other experiments. Fig. 2. View largeDownload slide Effects of UVB radiation on development of Tetranychus urticae larvae at (a, b) 25°C and (c) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Fig. 2. View largeDownload slide Effects of UVB radiation on development of Tetranychus urticae larvae at (a, b) 25°C and (c) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. The survival rate of adult females that developed under UVB radiation at 0.025 W m−2 (94.7%, 95% CI = 86.2–98.3%) was not significantly different from those developing without UVB irradiation (0 kJ m−2 d−1; 85.3%, 95% CI = 74.8–92.1%; Fisher’s exact test, P = 0.1) for 5 d after the first oviposition. Two females escaped without egg production in the second replication with no UVB radiation, so we removed these females from the analysis of egg production. Egg production was not significantly affected by UVB radiation; the number of eggs produced by females developing under UVB radiation was 28.9 ± 1.5 (SE) and that by females developed without UVB irradiation was 25.2 ± 1.5 (SE; nested ANOVA, UVB effects: P = 0.09, UVB × replication: P = 0.676). Effects of Daily UVB Irradiation and Temperature on Egg Hatchability of N. californicus Egg hatchability of N. californicus was high even if exposed to UVB radiation. The hatchability of eggs irradiated with 0.27 kJ m−2 d−1 was 89.2 and 94.0% at 25 and 20°C, respectively, and not significantly different from that of eggs without UVB irradiation (P = 0.192 and P = 1 at 25 and 20°C, respectively; Fig. 3). Fig. 3. View largeDownload slide Effects of UVB radiation on the hatchability of Neoseiulus californicus eggs at (a) 25°C and (b) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Fig. 3. View largeDownload slide Effects of UVB radiation on the hatchability of Neoseiulus californicus eggs at (a) 25°C and (b) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Effects of Daily UVB Irradiation to Eggs and Juveniles on the Development of N. californicus Most larva developed to adults without UVB exposure during the egg to adult stage (egg/larval stage: −/−), while the highest mortality was detected in the individuals irradiated during both egg and juvenile stages (+/+; Fig. 4). The next highest mortality was detected in the individuals irradiated only at the egg stage (+/−) and the third highest was in those irradiated only during the juvenile stages after hatching (−/+). The results of the statistical analysis revealed that UVB irradiation on eggs (two-way ANOVA, P = 9.58 × 10−4) and juveniles (P = 0.0124) affected developmental success, whereas, no interaction was detected between UVB irradiation on eggs and juveniles (P = 0.636). Fig. 4. View largeDownload slide Effects of UVB radiation at egg and juvenile stages from larva to adult on development of Neoseiulus californicus at 25°C. ‘−’ and ‘+’ indicate UVB irradiation at 0 and 0.27 kJ m−2 d−1 at egg/larval stage, respectively. Vertical lines on bars show SEs. Fig. 4. View largeDownload slide Effects of UVB radiation at egg and juvenile stages from larva to adult on development of Neoseiulus californicus at 25°C. ‘−’ and ‘+’ indicate UVB irradiation at 0 and 0.27 kJ m−2 d−1 at egg/larval stage, respectively. Vertical lines on bars show SEs. Photoreactivation in N. californicus Eggs In the dark control, eggs were seriously damaged by a single acute UVB radiation at 0.29 kJ m−2, and the hatchability was reduced to 16.7% and 31.7% in N. californicus reared on T. urticae and P. citri, respectively (Fig. 5a). However, the hatchability recovered to 94.2% and 89.7% in N. californicus reared on T. urticae and P. citri, respectively, by subsequent VIS irradiation at 365.6 kJ m−2. GLM analysis revealed that VIS irradiation showed the largest positive value of ‘Estimate’, meaning that VIS had the largest effect on increased hatchability (Table 1a; P = 4 × 10−15). The negative ‘Estimate’ of Tu (reared on T. urticae) indicated that prey P. citri increased hatchability of N. californicus eggs in comparison with prey T. urticae (P = 0.011). Statistical significance was also detected in the interaction between Tu and VIS (P = 0.0176), indicating that photoreactivaion was more effective in the eggs of N. californicus reared on T. urticae than P. citri. Fig. 5. View largeDownload slide Effects of photoreactivation on (a) egg hatchability and (b) survival of hatched larvae in the Neoseiulus californicus reared on Tetranychus urticae (Tu) and Panonychus citri (Pc). Gray and white bars represent dark control and photoreactivation treatment, respectively. Vertical lines on bars show 95% CIs. Fig. 5. View largeDownload slide Effects of photoreactivation on (a) egg hatchability and (b) survival of hatched larvae in the Neoseiulus californicus reared on Tetranychus urticae (Tu) and Panonychus citri (Pc). Gray and white bars represent dark control and photoreactivation treatment, respectively. Vertical lines on bars show 95% CIs. Table 1. Analyses of the effects of photoreactivation on egg hatchability (a) and survival of hatched larvae (b) of Neoseiulus californicus by generalized linear model (GLM, family = binomial) Factora  Estimate  SE  z-value  P-value  (a) Egg hatchability   (Intercept)  −0.7691  0.1962  −3.919  9 × 10−5   Tu  −0.8403  0.3303  −2.544  0.011   VIS  2.9356  0.3739  7.850  4 × 10−15   Tu × VIS  1.4772  0.6222  2.374  0.0176  (b) Survival of hatched larvae   (Intercept)  0.8979  0.3577  2.510  0.0121   Tu  −2.4384  0.7299  −3.341  0.0008   VIS  1.5000  0.5141  2.918  0.0035   Tu × VIS  2.4719  0.8973  2.755  0.0058  Factora  Estimate  SE  z-value  P-value  (a) Egg hatchability   (Intercept)  −0.7691  0.1962  −3.919  9 × 10−5   Tu  −0.8403  0.3303  −2.544  0.011   VIS  2.9356  0.3739  7.850  4 × 10−15   Tu × VIS  1.4772  0.6222  2.374  0.0176  (b) Survival of hatched larvae   (Intercept)  0.8979  0.3577  2.510  0.0121   Tu  −2.4384  0.7299  −3.341  0.0008   VIS  1.5000  0.5141  2.918  0.0035   Tu × VIS  2.4719  0.8973  2.755  0.0058  aTu: prey on T. urticae, VIS: visible light irradiation after UVB irradiation. View Large Most N. californicus larvae that had hatched from reactivated eggs by VIS irradiation survived at least just after hatching (day 4), whereas the survival rate of larvae hatched from eggs reared on T. urticae without photoreactivation by VIS was markedly reduced (Fig. 5b). The survival rate of N. californicus larvae hatched from eggs reared on P. citri without photoreactivation by VIS (dark control) also decreased, but it remained at 71.1%. Consequently, photoreactivation at egg stage increased the survival rate of hatched larvae (GLM, P = 0.0035) and rearing on P. citri increased the efficiency of photoreactivation at the egg stage on the survival rate of hatched larvae in comparison with rearing on T. urticae (P = 0.0008; Table 1b). The interaction between prey species and photoreactivation by VIS was also statistically significant (P = 0.0058), and the large positive ‘Estimate’ (2.4719) indicated that the effect of photoreactivation at the egg stage on the survival rate of hatched larvae was also more effective in N. californicus reared on T. urticae than that on P. citri. Discussion The marked recovery from UVB damage suggests that photoreactivation may be a major function for spider mites in surviving ambient UVB radiation. In this study, we kept the mites under dark conditions for 5 h from the end of UVB irradiation, so that the photoreactivation system of T. urticae eggs was likely disabled. In Murata and Osakabe (2014), hatchability of T. urticae eggs irradiated with UVB of 0.29 kJ m−2 at 25°C without photoreactivation was 14.9%, and that reactivated by VIS irradiation after 4-h time lag was 17.4%, whereas 68.1% of eggs hatched when they were reactivated with VIS without time lag after UVB irradiation. In this study, no egg hatched at 0.27 kJ m−2 d−1 at 25°C. The LD50 value of T. urticae eggs was reported as 0.58 kJ m−2 at 25°C (Murata and Osakabe 2013). The LD50 value was higher in T. urticae larvae (1.19 kJ m−2) than eggs (Murata and Osakabe 2013), and they could be reactivated by VIS irradiation even with a 4-h time lag (Murata and Osakabe 2014). Nevertheless, most larvae died under UVB irradiation at 0.59 kJ m−2 d−1 in this study. Although we did not have information about damage accumulation by such intermittent UVB irradiation, the results suggest the improvement of control effects on spider mite eggs by daily irradiation in the greenhouse and also supports the significance of a UVB irradiation time at midnight, making a sufficient time lag between UVB irradiation and sunrise. Although the effects of UVB irradiation at 0.13 kJ m−2 on T. urticae eggs was minimal at 25°C, a conspicuous increase in the deleterious effect of this dose was observed at 20°C; indeed, no egg hatched. A similar trend, that UVB vulnerability was higher at the lower temperature, was also found in T. urticae larvae exposed to 0.27 kJ m−2 d−1; survival to adulthood decreased, from 84.6% at 25°C to 26.4% at 20°C. This suggests that we could favorably reduce UVB dose in the winter when sunscald risk increases to occur on strawberry leaves. Sakai et al. (2012) reported marked degradation of hatchability in T. urticae eggs in spring in comparison with summer and autumn. Their results suggested that this was due to the slower development under lower temperature and the relatively higher UVB radiation in spring because hatchability was negatively correlated with cumulative UVB dose over the egg periods (Sakai et al. 2012). Reciprocity between UVB intensity and irradiation periods in UVB damage was also demonstrated by Murata and Osakabe (2013). The relationship between developmental rates and temperature is common to poikilothermic arthropods. Nevertheless, the effects of temperature on survival and photoreactivation from UVB damage are controversial in Daphnia species; some species are vulnerable to UVB damage under low temperature versus higher temperature but the opposite is true in other species (Williamson et al. 2002, Macfadyen et al. 2004, Connelly et al. 2009). This suggests variability in responses of protection systems involving photoreactivation and perhaps antioxidant activity to temperature among species. Unlike T. urticae, hatchability of N. californicus eggs was unaffected by UVB irradiation at 0.27 kJ m−2 d−1 at both 25°C and 20°C. Hatchability of N. californicus eggs under intermittent UVB irradiation at 0.27 kJ m−2 d−1, 25°C (89.2%) was markedly higher than after a single acute UVB irradiation at 0.28 kJ m−2 at 25°C (23.1%) in Tachi and Osakabe (2012). In our photoreactivation experiments, hatchability of N. californicus eggs reared on T. urticae and irradiated with UVB at 0.29 kJ m−2 at 25°C without VIS showed 16.7%, corresponding to Tachi and Osakabe (2012), whereas in the eggs irradiated with UVB followed by VIS irradiation, hatchability recovered, to 94.3%, corresponding to the results with intermittent UVB irradiation to N. californicus. Although N. californicus eggs were placed in a laboratory illuminated with fluorescent lights after UVB irradiation in Tachi and Osakabe (2012), the efficiency of photoreactivation depended on cumulative VIS irradiance (Murata and Osakabe 2014), so that photoreactivation may have been minimal in Tachi and Osakabe (2012) due to weaker illumination in the laboratory. Moreover, Murata and Osakabe (2014) calculated the maximum capacity of photoreactivation to be up to 57% of the eggs reactivated from UVB damage in T. urticae. This suggested that N. californicus possessed a prominent capacity for photoreactivation. In contrast, Koveos et al. (2017) tested the photoreactivation capacity of phytoseiid mites using concurrent UVB and white light radiation and concluded that spider mites relied on a photoreactivation system to recover from UVB damage but phytoseiid mites did not. Connelly et al. (2009) suggested the possibility that the difference in experimental conditions between concurrent UVB and VIS radiation and a single acute UVB radiation following VIS radiation caused variation in the response of Daphnia species to high and low temperatures. There is a need to determine whether photoreactivation would still work under intermittent UVB radiation in phytoseiid mites. Although the hatchability under intermittent UVB irradiation was higher in N. californicus than T. urticae, the residual effects of UVB irradiation to eggs on juvenile survival was found in N. californicus reared on T. urticae, and UVB irradiation to the larvae and successive juvenile stages also decreased development to adulthood, as well as that in T. urticae. Consequently, half of the larvae died in development when both eggs and juveniles were reared under intermittent UVB irradiation at 0.27 kJ m−2 at 25°C. A residual effect of UVB irradiation to eggs was also detected in the dark control for the photoreactivation experiment with a single acute UVB irradiation; many larvae reared on T. urticae died immediately after hatching, with the result that only 17.6% of hatched larvae survived just after hatching. Spider mites exposed to UVB radiation at lethal doses died primarily at sensitive developmental stages, such as embryo development and the chrysalis stages before molting (Murata and Osakabe 2014) and photoreactivation may be a key factor determining survivorship, whereas the residual effects of UVB radiation have not been reported before in spider mites. The residual effects indicated divergence in a key factor for phytoseiid mites to survive UVB radiation. In contrast, the egg hatchability and the survival rate of hatched larvae in photoreactivation experiments improved to 31.7% and 71.1% in N. californicus reared on P. citri, respectively. Many plant-dwelling mites avoid solar UVB radiation by remaining on lower leaf surfaces of host plants (Ohtsuka and Osakabe 2009; Sudo and Osakabe 2011, 2015). In contrast, P. citri is more tolerant to UVB radiation than T. urticae and resides not only on the lower leaf surfaces but also on upper leaf surfaces (Fukaya et al. 2013). P. citri possesses astaxanthin as a major pigment (Metcalf and Newell 1962, Atarashi et al. 2017), which is a prominent antioxidant among the carotenoids (Miki 1991, Kobayashi and Sakamoto 1999). Atarashi et al. (2017) showed that astaxanthin reduced the accumulation of lipid peroxide (LPO) caused by UVB- and high-temperature-generated reactive oxygen species (ROS) in P. citri. Thermal stress enhances ROS generation and LPO accumulation due to a deficiency in enzymatic antioxidant systems (catalase, superoxide dismutase, peroxidase, and glutathione S-transferase) in a phytoseiid mite Neoseiulus cucumeris (Oudemans) (Acari: Phytoseiidae), with the result that the mite died (Zhang et al. 2014). A similar deficiency in enzymatic antioxidant systems was also detected in P. citri, so that the presence of a non-enzymatic defense system was suggested (Yang et al. 2010). T. urticae does not accumulate astaxanthin in the summer form, whereas they accumulate keto-carotenoids, including astaxanthin, in the diapausing winter form (Veerman 1974, Kawaguchi et al. 2016) and increase UVB tolerance (Suzuki et al. 2009). This indicates that astaxanthin in P. citri was possibly responsible for the improvement in the hatchability of eggs and survival of hatched larvae of N. californicus. Variation in the effects of temperature on the biological impact of intermittent UVB irradiation between spider mites and phytoseiid mites may be valuable to develop appropriate irradiation conditions for an IPM strategy. Major biological damage suffered from UVB radiation is product of DNA lesions, such as cyclobutane pyrimidine dimers and pyrimidine (6–4) pyrimidone photoproducts and the generation of ROS, such as singlet oxygen (Sinha and Häder 2002). Although the impact of ROS is not necessarily lethal in spider mites (Atarashi et al. 2017), a non-enzymatic antioxidant system may be more significant in surviving UVB radiation in phytoseiid mites and their UVB tolerance may be improved by food advantageously together with the practical use of UVB lamps. The serious development of acaricide resistance in spider mites causes failure of chemical control. The combined use of UVB lamps and light reflection sheets is a novel physical control method that enables to control spider mite and powdery mildew simultaneously in strawberry greenhouse and likely to be effective also on acaricide resistant mites. However, leaf sunscald in winter by UVB irradiation is a problem for producers, and many are concerned about the mal-effects on phytoseiid mites by the UVB radiation. Our findings indicate that it will be possible to reduce UVB dose while maintaining the control effects on spider mites in winter and to use phytoseiid mites concurrently with UVB irradiation, contributing IPM strategies in strawberry greenhouse. Supplementary Material Supplementary data are available at Environmental Entomology online. Acknowledgments This work was supported by Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), ‘Technologies for creating next-generation agriculture, forestry and fisheries’ (funding agency: Bio-oriented Technology Research Advancement Institution, NARO) and JSPS KAKENHI (a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan) grant number 26292029. References Cited Atarashi, M., Manabe Y., Kishimoto H., Sugawara T., and Osakabe M.. 2017. Antioxidant protection by astaxanthin in the citrus red mite (Acari: Tetranychidae). 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Google Scholar CrossRef Search ADS PubMed  Zhang, G. H., Liu H., Wang J. J., and Wang Z. Y.. 2014. Effects of thermal stress on lipid peroxidation and antioxidant enzyme activities of the predatory mite, Neoseiulus cucumeris (Acari: Phytoseiidae). Exp. Appl. Acarol . 64: 73– 85. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Entomology Oxford University Press

Effects of Low Temperature on Spider Mite Control by Intermittent Ultraviolet-B Irradiation for Practical Use in Greenhouse Strawberries

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Abstract

Abstract The application of ultraviolet-B (UVB) radiation to control spider mites is challenging as a key technology for integrated pest management (IPM) in greenhouse strawberries in Japan. To address this, concurrent use of phytoseiid mites and reduced UVB irradiance is desirable to ensure control effects in areas shaded from UVB radiation and to minimize the sunscald in winter, respectively. We designed experiments reproducing the UVB dose on the lower leaf surfaces in strawberry and evaluated the effects of intermittent UVB irradiation at midnight for practical application in the greenhouse and low temperature on the survival of the spider mite Tetranychus urticae Koch (Acari: Tetranychidae) and damage to the phytoseiid mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae). The midnight intermittent UVB irradiation effectively suppressed egg hatching and development of larvae of T. urticae, and the control effect was reinforced at 20°C (no eggs hatched at 0.13 kJ m−2 d−1) rather than, at 25°C (70.8% eggs hatched). In contrast, the hatchability of N. californicus eggs was unaffected by intermittent UVB irradiation at 0.27 kJ m−2 d−1 at 25°C and 20°C. However, residual effects of UVB irradiation to N. californicus eggs on survival of hatched larvae were seen, so that reducing the UVB dose is also advantageous for this phytoseiid mite. N. californicus showed a photoreactivation capacity, whereas their UVB tolerance was improved by prey species, suggesting the possibility of the improvement of phytoseiid mites by diet. The reduction of UVB dose and concurrent use of phytoseiid mites increase reliability of the UVB method in IPM strategies in strawberry greenhouse. Deleterious effects of ultraviolet-B (UVB; 280–315 nm) radiation on spider mites have been reported over the last decade (Suzuki et al. 2009, Ohtsuka and Osakabe 2009, Sakai et al. 2012, Murata and Osakabe 2017). A UVB lamp was developed to control powdery mildew in greenhouses (Kanto et al. 2009, 2014; Kobayashi et al. 2013). Conspicuous control effects on spider mite populations by the combined use of UVB lamps and light reflection sheets were demonstrated in melons (Masui et al. 2013) and strawberries (Tanaka et al. 2016) in greenhouses. Spider mites and powdery mildew are economically important pest and disease, respectively, especially in strawberries, so that simultaneous control of these pest and disease is required. Moreover, the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae), a major mite pest in strawberry, has developed serious resistance to the most acaricides (Van Leeuwen et al. 2010, 2015). A control method which can simultaneously control spider mites and powdery mildew may be a good candidate for integrated pest management (IPM) in greenhouses, especially for strawberry production. Tanaka et al. (2016) used wide intervals of rows (0.4–0.5 m) and plants (0.3–0.35 m) to make it easier for UVB radiation to reach lower leaf surfaces where spider mites reside. In contrast, under economically favorable narrower row- (0.25 m) and plant-interval (0.23–0.25 m) conditions, leaves may become overgrown, increasing the region of leaves shaded from UVB radiation. Increasing UVB intensity may decrease the shaded area on overgrown leaves. However, excessive UVB irradiation may increase plant damage such as sunscald occurred in strawberry leaves in winter (Tanaka et al. 2016). In practical terms and for effective IPM, concurrent use of phytoseiid mites with UVB and reduction in UVB irradiance to minimize the sunscald in winter are desirable. Studies indicated high vulnerability to UVB radiation in phytoseiid mites (Onzo et al. 2010, Ghazy et al. 2016); their prompt avoidance from areas irradiated with UVB has also been suggested (Tachi and Osakabe 2012, 2014). In these studies, vulnerability was tested using a single acute irradiation of UVB. Unlike these, UVB irradiation was performed for 3 h at midnight in strawberry greenhouses (Tanaka et al. 2016). Spider mites are potentially reactivated by photo-enzymatic repair (PER) of DNA lesions, using energy from visible light (VIS) and ultraviolet-A (UVA; 315–400) in sunshine during the day under greenhouse conditions (Suzuki et al. 2014; Murata and Osakabe 2014, 2017). However, the effects of photoreactivation were reduced with increasing time lag between UVB irradiation and exposure to VIS in eggs, and no reactivation effects remained when the time lag was 4 h (Murata and Osakabe 2014). For that reason Tanaka et al. (2016) irradiated strawberries with UVB at midnight and ended 3–4 h before sunrise, whereas the 4-h time lag did not interfere with the photoreactivation of larvae (Murata and Osakabe 2014). Thus, this method may be effective suppressing egg hatching while less effective in interfering with juvenile development of spider mites. In phytoseiid mites, Koveos et al. (2017) tested the effects of photoreactivation under simultaneous UVB and white light radiation. Unlike T. urticae, the white light radiation did not increase egg hatchability of phytoseiid mites, indicating that the phytoseiid mites were more sensitive to UVB damage than spider mites and did not rely on a photoreactivation system (Koveos et al. 2017). Despite demand to reduce UVB irradiance in winter, the effects of low temperature on survival and DNA repair in mites exposed to UVB radiation are largely unknown. According to studies in other organisms, the effects of temperature on damage and repair are unlikely to be simple responses. Survival and photoreactivation rates of freshwater Daphnia species (Cladocera, Daphiidae) were higher at 10°C than at 20°C (Connelly et al. 2009). Similarly, the yield of DNA damage after exposure to UVB radiation at 12°C is lower than that at 24°C in a marine bacterium Shingopyxis alaskensis (Matallana-Surget et al. 2010). In contrast, Escherichia coli cells grown at 20°C are more sensitive to UVB radiation than those grown at 37°C (Mangoli et al. 2014). Nevertheless, there are reports of higher vulnerability to UVB radiation of Daphnia species at lower temperatures (Williamson et al. 2002, Macfadyen et al. 2004). In this study, we designed experiments reproduced the UVB dose on the lower leaf surfaces in strawberry greenhouse in a chamber and evaluated the effects of intermittent irradiation of UVB (3-h irradiation every night) and that of lower temperature (20°C) on the survival of T. urticae and the phytoseiid mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) in comparison with a higher temperature (25°C). We also tested the capacity for photoreactivation in phytoseiid mites. Materials and Methods Mites T. urticae and N. californicus were used for the evaluation of biological impact of UVB radiation, simulating the irradiation conditions in a greenhouse (Tanaka et al. 2016). A T. urticae population was obtained from several areas in Japan and was reared on kidney bean Phaseolus vulgaris L. leaves. The N. californicus strain had been originally collected from Japanese pear Pyrus pyrifolia (Burm.f.) Nakai trees in Matsukawa, Nagano Prefecture, Japan (35° 36ʹ N, 137° 55ʹ E) in September 2000 and was reared on the T. urticae population in the laboratory at 25°C (approximately 80% RH) under a photoperiod of 16:8 (L:D) h. In the experiments on photoreactivation, N. californicus was reared on both T. urticae and the citrus red mite Panonychus citri (McGregor) (Acari: Tetranychidae) for more than two generations prior to use. The P. citri population was collected from Satsuma mandarin Citrus unshiu Marc. (‘Tsunoka’) at the National Institute of Fruit Tree Science, Minamishimabara, Nagasaki, Japan (32° 36ʹ N, 130° 11ʹ E) on 1 June 2007 and reared on kidney bean leaves in the laboratory. All kidney bean leaves used for the rearing cultures and experiments were primary leaves. UVB and VIS Irradiation Conditions The UVB irradiance was measured using an irradiance meter (X11) equipped with a UV-3702–4 detector head (Gigahertz-Optik, Türkenfeld, Germany). The VIS irradiance in the photoreactivation experiment was measured using a photo-radiometer (HD2102.2) equipped with an irradiance measurement probe (LP 471 RAD; Delta OHM, Padova, Italy). Irradiation equipment was similar to that used by Murata and Osakabe (2014, 2017). A UVB lamp (6 W; Panasonic Electric Works, Osaka, Japan) was affixed from a shelf 67 cm overhead in a growth chamber at 25°C and 20°C. The growth chamber was illuminated with fluorescent lights (≈7,000 lx on the shelf) from 08:00 to 22:00 (simulated daytime; 14:10 [L:D] h) every day. The UVB lamp was turned on at 00:00 and turned off at 03:00 every day in the simulated nighttime. The UVB intensity of direct irradiation was 0.16 W m−2 on the shelf. The UVB lamp was covered with polyethylene sheets except during the photoreactivation experiment. Polyethylene sheets have no effects on the wavelength spectrum of UVB irradiation, and the intensity was adjusted to 0.012, 0.025, and 0.055 W m−2: UVB irradiances were 0.13, 0.27, and 0.59 kJ m−2 d−1, respectively. The UVB intensities of 0.025 and 0.055 W m−2 roughly corresponded with that on lower leaf surfaces of greenhouse strawberries (0.022–0.053 W m−2) in Tanaka et al. (2016). The daily cumulative UVB irradiances were equivalent to or below the LD50 value for T. urticae eggs (0.58 kJ m−2) reported by Murata and Osakabe (2013). A single acute UVB irradiation was performed with direct irradiation from the UVB lamp in the photoreactivation experiments. During photoreactivation experiments, the fluorescent lights were turned off; and, two halogen lamps (130 W; JDR110V-85WHM/K7-H; Ushio Lighting, Tokyo, Japan; set at an interval of 22.5 cm) affixed from a shelf 67 cm overhead in the dark growth chamber were used as VIS sources; the irradiance value was 67.7 W m−2 on the shelf. Effects of Daily UVB Irradiation and Temperature on T. urticae Egg Hatchability We prepared two Petri dishes (9 cm in diameter), one for UVB treatment and the other for non-irradiated control. Four pieces of squared kidney bean leaves (2 × 2 cm) were placed on water-soaked cotton in a Petri dish. Five adult T. urticae females were introduced to each kidney bean leaf. One Petri dish was placed in a plastic basket (20 × 27 × 8.5 cm) in which the top was covered with UV-opaque film (HB3 polyester film; DuPont Teijin Films, Chester, VA) (UV−) and the other Petri dish was placed in the same plastic basket covered with UVB-transparent film (polyvinylidene chloride film; Saran Wrap; Asahi Kasei Home Products Co., Tokyo, Japan) (UV+) and allowed to lay eggs in the growth chamber. The UV-opaque film filtered out more than 90% of UV at wavelengths less than 380 nm (Sakai and Osakabe 2010; Supplementary Fig. 1), whereas the UVB-transparent film transmitted more than 90% of UVB and VIS (Supplementary Fig. 1). Humidity was not adjusted and fluctuated between 45 and 65% RH in the plastic basket. After 24 h the adult females were removed and the numbers of eggs on the leaves were arranged to 20 (80 eggs per Petri dish; day 0). The eggs were kept in the same growth chamber and the number of hatched larvae and unhatched eggs were counted 6 d later (day 6). Experiments were performed for UVB irradiance of 0.13 and 0.27 kJ m−2 d−1 at 25°C and 0.13 kJ m−2 d−1 at 20°C. All experiments were replicated three times. Data sets were pooled over three replications. Variation of hatchability among treatments (0, 0.13, and 0.27 kJ m−2 d−1) at 25°C was analyzed with Fisher’s exact test using the ‘pairwise.fisher.test’ module of the ‘fmsb’ package and the ‘fisher.test’ module, in the ‘R’ software (ver. 3.3.3; R Core Team 2017). Survival of Larvae Two Petri dishes were prepared as described for T. urticae egg hatchability experiments, but the number of leaves per Petri dish was two at 20°C. Twenty T. urticae larvae that were within 3 h after hatching were introduced to each leaf (80 and 40 larvae per Petri dish for the experiments at 25°C and 20°C, respectively). The larvae were prepared by a method to synchronize egg hatching, as described in Ubara and Osakabe (2015), without UVB radiation. The Petri dishes were set in the plastic basket for UV− and UV+ and reared in the growth chamber until adulthood, death, or escape. Escaped individuals were omitted from the following data analysis. Experiments were performed for UVB irradiances of 0.27 and 0.55 kJ m−2 d−1 at 25°C and 0.27 kJ m−2 d−1 at 20°C. In the experiments with 0.27 kJ m−2 d−1 UVB at 25°C, we checked egg production by adult females randomly chosen from survivors (15 ♀♀ in the first replication and 30 ♀♀ in the second and third replications) individually reared on kidney bean leaves for 5 d after the first oviposition. All experiments were replicated three times. We pooled the data sets of the survival of larvae to adults and developed adult females over the replications. The difference of the survival between treatments with and without UVB irradiation was analyzed by Fisher’s exact test using the ‘fisher.test’ module in the R software. The effects of UVB radiation on egg production were evaluated by nested analysis of variance (nested ANOVA) after Bartlett’s test using the ‘aov’ and ‘bartlett.test’ modules in the R software. Effects of Daily UVB Irradiation and Temperature on Egg Hatchability of N. californicus We prepared two and three Petri dishes for the experiments at 25°C and 20°C, respectively. Each Petri dish contained a squared kidney bean leaf (4 × 4 cm), placed on water-soaked cotton. Ten adult female T. urticae were introduced to each leaf. The Petri dishes were set in the plastic baskets for UV− (two of three dishes were assigned at 20°C) and UV+ and placed in the growth chamber where irradiated with UVB at 0.025 W m−2 for 3 h (0.27 kJ m−2 d−1) every night. After 24 h, when T. urticae females had laid eggs, 10 adult females of N. californicus were introduced to each leaf and reared together with T. urticae females and their eggs for 24 h in the same chamber. Then, females of T. urticae and N. californicus were removed. Eggs of N. californicus laid on the leaf were continuously reared in the same chamber (day 0), and their hatchability was determined 3 d later (day 3) and 5 d later (day 5) in the experiments at 25°C and 20°C, respectively. All experiments were replicated three times. The numbers of eggs used for experiments were 65 (20–25 eggs per replication) and 72 (22–25) at 25°C and 67 (20–27) and 113 (33–40) at 20°C. The data sets over three replications were pooled, and differences in the hatchability between treatments with and without UVB irradiation were analyzed by Fisher’s exact test using the ‘fisher.test’ module in the R software. Effects of Daily UVB Irradiation at Egg Stage and After Hatching on Development of N. californicus Four Petri dishes were prepared in the same manner as that in the experiments on the hatchability of N. californicus eggs. Ten adult females of T. urticae were introduced to each leaf and two Petri dishes were set in the plastic basket for UV−, and the remaining two were in that for UV+ and placed in the growth chamber at 0.025 W m−2 for 3 h (0.27 kJ m−2 d−1) UVB irradiation at 25°C. After 24 h, 10 adult females of N. californicus were introduced to each leaf and allowed to lay eggs. The N. californicus females were removed from leaves. The Petri dishes containing N. californicus eggs and eggs and adult females of T. urticae on the leaves were kept continuously in baskets in the chamber. Three days later, unhatched N. californicus eggs were removed from leaves. Then, one of two Petri dishes in the UV− basket and one of two Petri dishes in the UV+ baskets were interchanged (day 0). The N. californicus larvae were reared until adult emergence. Developmental success was determined on day 4. By this method, we set four treatments: 1) irradiated with UVB from egg to adult (n = 17–21 [in each replication]); 2) irradiated from larva to adult but not irradiated during egg and the beginning of larval stage (n = 17–20); 3) irradiated during egg and the beginning of the larval stage, but not irradiated from larva to adult (n = 15–25); and 4) non-irradiated during egg to adult (n = 16–20; control). All experiments were replicated three times. Prior to data analysis, we performed an arc sine transformation and Bartlett’s test using the ‘bartlett.test’ module, then two-way ANOVA using the ‘aov’ module in the R software. Because unhatched eggs were excluded from the data analysis, we could evaluate the residual effects of UVB irradiation to eggs and direct effects of UVB irradiation on the juvenile stages of development. Photoreactivation in N. californicus Eggs Two Petri dishes were containing four pieces of squared kidney bean leaves (2 × 2 cm) on water-soaked cotton. Ten T. urticae adult females or 40 P. citri females were introduced to each leaf and placed in a laboratory at 25°C under a photoperiod of 16:8 (L:D) h. The spider mite females were removed after 24 h. The five adult females of N. californicus reared on T. urticae and P. citri were introduced to the leaves containing T. urticae eggs and P. citri eggs, respectively, and allowed to lay eggs for 24 h. Then, we removed phytoseiid mite females and immediately exposed their eggs to UVB radiation at 0.16 W m−2 for 30 min (0.29 kJ m−2) in the growth chamber. Immediately after the end of UVB irradiation, one Petri dish was put inside a cardboard box (dark box: 24.0 × 16.5 × 10.8 cm). The dark box containing a Petri dish (dark control) and the other Petri dish (photoreactivation treatment) were exposed to VIS (halogen lamp, 67.7 W m−2) for 90 min (365.6 kJ m−2). Then, the Petri dish for photoreactivation treatment was also put inside the cardboard box together with the dark control and placed in the laboratory (day 0). Eggs were observed every day, although the dark condition was interrupted briefly by the observation. Egg hatchability was determined 4 d later (day 4). Larvae that died soon after hatching were also counted on day 4, and used to calculate survival rates. All experiments were replicated twice. The numbers of N. californicus eggs reared on T. urticae were 52 and 50 for the dark control and 52 and 53 for photoreactivation treatment in the first and second replications, respectively; the numbers of N. californicus eggs reared on P. citri were 64 and 56 for dark control and 53 and 54 for the photoreactivation treatment. Effects of VIS irradiation after UVB irradiation on egg hatchability and survival of hatched larvae of N. californicus were evaluated by a generalized linear model (GLM; family = binomial) using the ‘glm’ module in the R software. Results Effects of Daily UVB Irradiation and Temperature on T. urticae Egg Hatchability The hatchability of eggs exposed to no UVB radiation (0 kJ m−2 d−1; UV−) was 91.3% and 92.1% at 25°C and 20°C, respectively (Fig. 1). In contrast, no egg hatched with UVB irradiation at 0.025 W m−2 (0.27 kJ m−2 d−1) at 25°C (Fisher’s exact test, P = 2 × 10−16; Fig. 1a). With UVB irradiation at 0.012 W m−2 (0.13 kJ m−2 d−1), egg hatchability decreased to 70.8% at 25°C (P = 1.2 × 10−8; Fig. 1a) and to zero at 20°C (P = 2.2 × 10−16; Fig. 1b). Fig. 1. View largeDownload slide Effects of UVB radiation on the hatchability of Tetranychus urticae eggs at (a) 25°C and (b) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Fig. 1. View largeDownload slide Effects of UVB radiation on the hatchability of Tetranychus urticae eggs at (a) 25°C and (b) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Survival of Larvae More than 94% of larvae exposed to no UVB radiation (0 kJ m−2 d−1; UV−) developed to adults at 25°C and 87.5% at 20°C (Fig. 2). Most larvae died with UVB irradiation at 0.055 W m−2 (0.59 kJ m−2 d−1) at 25°C (Fisher’s exact test, P = 2.2 × 10−16; Fig. 2b). With UVB irradiation at 0.025 W m−2 (0.27 kJ m−2 d−1), development was decreased slightly, to 84.6%, at 25°C (P = 1.93 × 10−6; Fig. 2a) and reduced significantly, to 36.7%, at 20°C (P = 1.87 × 10−11; Fig. 2c). Only one individual irradiated with UVB escaped from the leaf in the development experiment at 20°C; however, no individuals escaped in the other experiments. Fig. 2. View largeDownload slide Effects of UVB radiation on development of Tetranychus urticae larvae at (a, b) 25°C and (c) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Fig. 2. View largeDownload slide Effects of UVB radiation on development of Tetranychus urticae larvae at (a, b) 25°C and (c) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. The survival rate of adult females that developed under UVB radiation at 0.025 W m−2 (94.7%, 95% CI = 86.2–98.3%) was not significantly different from those developing without UVB irradiation (0 kJ m−2 d−1; 85.3%, 95% CI = 74.8–92.1%; Fisher’s exact test, P = 0.1) for 5 d after the first oviposition. Two females escaped without egg production in the second replication with no UVB radiation, so we removed these females from the analysis of egg production. Egg production was not significantly affected by UVB radiation; the number of eggs produced by females developing under UVB radiation was 28.9 ± 1.5 (SE) and that by females developed without UVB irradiation was 25.2 ± 1.5 (SE; nested ANOVA, UVB effects: P = 0.09, UVB × replication: P = 0.676). Effects of Daily UVB Irradiation and Temperature on Egg Hatchability of N. californicus Egg hatchability of N. californicus was high even if exposed to UVB radiation. The hatchability of eggs irradiated with 0.27 kJ m−2 d−1 was 89.2 and 94.0% at 25 and 20°C, respectively, and not significantly different from that of eggs without UVB irradiation (P = 0.192 and P = 1 at 25 and 20°C, respectively; Fig. 3). Fig. 3. View largeDownload slide Effects of UVB radiation on the hatchability of Neoseiulus californicus eggs at (a) 25°C and (b) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Fig. 3. View largeDownload slide Effects of UVB radiation on the hatchability of Neoseiulus californicus eggs at (a) 25°C and (b) 20°C. Vertical lines on bars show 95% CIs. P values were calculated using Fisher’s exact test. Effects of Daily UVB Irradiation to Eggs and Juveniles on the Development of N. californicus Most larva developed to adults without UVB exposure during the egg to adult stage (egg/larval stage: −/−), while the highest mortality was detected in the individuals irradiated during both egg and juvenile stages (+/+; Fig. 4). The next highest mortality was detected in the individuals irradiated only at the egg stage (+/−) and the third highest was in those irradiated only during the juvenile stages after hatching (−/+). The results of the statistical analysis revealed that UVB irradiation on eggs (two-way ANOVA, P = 9.58 × 10−4) and juveniles (P = 0.0124) affected developmental success, whereas, no interaction was detected between UVB irradiation on eggs and juveniles (P = 0.636). Fig. 4. View largeDownload slide Effects of UVB radiation at egg and juvenile stages from larva to adult on development of Neoseiulus californicus at 25°C. ‘−’ and ‘+’ indicate UVB irradiation at 0 and 0.27 kJ m−2 d−1 at egg/larval stage, respectively. Vertical lines on bars show SEs. Fig. 4. View largeDownload slide Effects of UVB radiation at egg and juvenile stages from larva to adult on development of Neoseiulus californicus at 25°C. ‘−’ and ‘+’ indicate UVB irradiation at 0 and 0.27 kJ m−2 d−1 at egg/larval stage, respectively. Vertical lines on bars show SEs. Photoreactivation in N. californicus Eggs In the dark control, eggs were seriously damaged by a single acute UVB radiation at 0.29 kJ m−2, and the hatchability was reduced to 16.7% and 31.7% in N. californicus reared on T. urticae and P. citri, respectively (Fig. 5a). However, the hatchability recovered to 94.2% and 89.7% in N. californicus reared on T. urticae and P. citri, respectively, by subsequent VIS irradiation at 365.6 kJ m−2. GLM analysis revealed that VIS irradiation showed the largest positive value of ‘Estimate’, meaning that VIS had the largest effect on increased hatchability (Table 1a; P = 4 × 10−15). The negative ‘Estimate’ of Tu (reared on T. urticae) indicated that prey P. citri increased hatchability of N. californicus eggs in comparison with prey T. urticae (P = 0.011). Statistical significance was also detected in the interaction between Tu and VIS (P = 0.0176), indicating that photoreactivaion was more effective in the eggs of N. californicus reared on T. urticae than P. citri. Fig. 5. View largeDownload slide Effects of photoreactivation on (a) egg hatchability and (b) survival of hatched larvae in the Neoseiulus californicus reared on Tetranychus urticae (Tu) and Panonychus citri (Pc). Gray and white bars represent dark control and photoreactivation treatment, respectively. Vertical lines on bars show 95% CIs. Fig. 5. View largeDownload slide Effects of photoreactivation on (a) egg hatchability and (b) survival of hatched larvae in the Neoseiulus californicus reared on Tetranychus urticae (Tu) and Panonychus citri (Pc). Gray and white bars represent dark control and photoreactivation treatment, respectively. Vertical lines on bars show 95% CIs. Table 1. Analyses of the effects of photoreactivation on egg hatchability (a) and survival of hatched larvae (b) of Neoseiulus californicus by generalized linear model (GLM, family = binomial) Factora  Estimate  SE  z-value  P-value  (a) Egg hatchability   (Intercept)  −0.7691  0.1962  −3.919  9 × 10−5   Tu  −0.8403  0.3303  −2.544  0.011   VIS  2.9356  0.3739  7.850  4 × 10−15   Tu × VIS  1.4772  0.6222  2.374  0.0176  (b) Survival of hatched larvae   (Intercept)  0.8979  0.3577  2.510  0.0121   Tu  −2.4384  0.7299  −3.341  0.0008   VIS  1.5000  0.5141  2.918  0.0035   Tu × VIS  2.4719  0.8973  2.755  0.0058  Factora  Estimate  SE  z-value  P-value  (a) Egg hatchability   (Intercept)  −0.7691  0.1962  −3.919  9 × 10−5   Tu  −0.8403  0.3303  −2.544  0.011   VIS  2.9356  0.3739  7.850  4 × 10−15   Tu × VIS  1.4772  0.6222  2.374  0.0176  (b) Survival of hatched larvae   (Intercept)  0.8979  0.3577  2.510  0.0121   Tu  −2.4384  0.7299  −3.341  0.0008   VIS  1.5000  0.5141  2.918  0.0035   Tu × VIS  2.4719  0.8973  2.755  0.0058  aTu: prey on T. urticae, VIS: visible light irradiation after UVB irradiation. View Large Most N. californicus larvae that had hatched from reactivated eggs by VIS irradiation survived at least just after hatching (day 4), whereas the survival rate of larvae hatched from eggs reared on T. urticae without photoreactivation by VIS was markedly reduced (Fig. 5b). The survival rate of N. californicus larvae hatched from eggs reared on P. citri without photoreactivation by VIS (dark control) also decreased, but it remained at 71.1%. Consequently, photoreactivation at egg stage increased the survival rate of hatched larvae (GLM, P = 0.0035) and rearing on P. citri increased the efficiency of photoreactivation at the egg stage on the survival rate of hatched larvae in comparison with rearing on T. urticae (P = 0.0008; Table 1b). The interaction between prey species and photoreactivation by VIS was also statistically significant (P = 0.0058), and the large positive ‘Estimate’ (2.4719) indicated that the effect of photoreactivation at the egg stage on the survival rate of hatched larvae was also more effective in N. californicus reared on T. urticae than that on P. citri. Discussion The marked recovery from UVB damage suggests that photoreactivation may be a major function for spider mites in surviving ambient UVB radiation. In this study, we kept the mites under dark conditions for 5 h from the end of UVB irradiation, so that the photoreactivation system of T. urticae eggs was likely disabled. In Murata and Osakabe (2014), hatchability of T. urticae eggs irradiated with UVB of 0.29 kJ m−2 at 25°C without photoreactivation was 14.9%, and that reactivated by VIS irradiation after 4-h time lag was 17.4%, whereas 68.1% of eggs hatched when they were reactivated with VIS without time lag after UVB irradiation. In this study, no egg hatched at 0.27 kJ m−2 d−1 at 25°C. The LD50 value of T. urticae eggs was reported as 0.58 kJ m−2 at 25°C (Murata and Osakabe 2013). The LD50 value was higher in T. urticae larvae (1.19 kJ m−2) than eggs (Murata and Osakabe 2013), and they could be reactivated by VIS irradiation even with a 4-h time lag (Murata and Osakabe 2014). Nevertheless, most larvae died under UVB irradiation at 0.59 kJ m−2 d−1 in this study. Although we did not have information about damage accumulation by such intermittent UVB irradiation, the results suggest the improvement of control effects on spider mite eggs by daily irradiation in the greenhouse and also supports the significance of a UVB irradiation time at midnight, making a sufficient time lag between UVB irradiation and sunrise. Although the effects of UVB irradiation at 0.13 kJ m−2 on T. urticae eggs was minimal at 25°C, a conspicuous increase in the deleterious effect of this dose was observed at 20°C; indeed, no egg hatched. A similar trend, that UVB vulnerability was higher at the lower temperature, was also found in T. urticae larvae exposed to 0.27 kJ m−2 d−1; survival to adulthood decreased, from 84.6% at 25°C to 26.4% at 20°C. This suggests that we could favorably reduce UVB dose in the winter when sunscald risk increases to occur on strawberry leaves. Sakai et al. (2012) reported marked degradation of hatchability in T. urticae eggs in spring in comparison with summer and autumn. Their results suggested that this was due to the slower development under lower temperature and the relatively higher UVB radiation in spring because hatchability was negatively correlated with cumulative UVB dose over the egg periods (Sakai et al. 2012). Reciprocity between UVB intensity and irradiation periods in UVB damage was also demonstrated by Murata and Osakabe (2013). The relationship between developmental rates and temperature is common to poikilothermic arthropods. Nevertheless, the effects of temperature on survival and photoreactivation from UVB damage are controversial in Daphnia species; some species are vulnerable to UVB damage under low temperature versus higher temperature but the opposite is true in other species (Williamson et al. 2002, Macfadyen et al. 2004, Connelly et al. 2009). This suggests variability in responses of protection systems involving photoreactivation and perhaps antioxidant activity to temperature among species. Unlike T. urticae, hatchability of N. californicus eggs was unaffected by UVB irradiation at 0.27 kJ m−2 d−1 at both 25°C and 20°C. Hatchability of N. californicus eggs under intermittent UVB irradiation at 0.27 kJ m−2 d−1, 25°C (89.2%) was markedly higher than after a single acute UVB irradiation at 0.28 kJ m−2 at 25°C (23.1%) in Tachi and Osakabe (2012). In our photoreactivation experiments, hatchability of N. californicus eggs reared on T. urticae and irradiated with UVB at 0.29 kJ m−2 at 25°C without VIS showed 16.7%, corresponding to Tachi and Osakabe (2012), whereas in the eggs irradiated with UVB followed by VIS irradiation, hatchability recovered, to 94.3%, corresponding to the results with intermittent UVB irradiation to N. californicus. Although N. californicus eggs were placed in a laboratory illuminated with fluorescent lights after UVB irradiation in Tachi and Osakabe (2012), the efficiency of photoreactivation depended on cumulative VIS irradiance (Murata and Osakabe 2014), so that photoreactivation may have been minimal in Tachi and Osakabe (2012) due to weaker illumination in the laboratory. Moreover, Murata and Osakabe (2014) calculated the maximum capacity of photoreactivation to be up to 57% of the eggs reactivated from UVB damage in T. urticae. This suggested that N. californicus possessed a prominent capacity for photoreactivation. In contrast, Koveos et al. (2017) tested the photoreactivation capacity of phytoseiid mites using concurrent UVB and white light radiation and concluded that spider mites relied on a photoreactivation system to recover from UVB damage but phytoseiid mites did not. Connelly et al. (2009) suggested the possibility that the difference in experimental conditions between concurrent UVB and VIS radiation and a single acute UVB radiation following VIS radiation caused variation in the response of Daphnia species to high and low temperatures. There is a need to determine whether photoreactivation would still work under intermittent UVB radiation in phytoseiid mites. Although the hatchability under intermittent UVB irradiation was higher in N. californicus than T. urticae, the residual effects of UVB irradiation to eggs on juvenile survival was found in N. californicus reared on T. urticae, and UVB irradiation to the larvae and successive juvenile stages also decreased development to adulthood, as well as that in T. urticae. Consequently, half of the larvae died in development when both eggs and juveniles were reared under intermittent UVB irradiation at 0.27 kJ m−2 at 25°C. A residual effect of UVB irradiation to eggs was also detected in the dark control for the photoreactivation experiment with a single acute UVB irradiation; many larvae reared on T. urticae died immediately after hatching, with the result that only 17.6% of hatched larvae survived just after hatching. Spider mites exposed to UVB radiation at lethal doses died primarily at sensitive developmental stages, such as embryo development and the chrysalis stages before molting (Murata and Osakabe 2014) and photoreactivation may be a key factor determining survivorship, whereas the residual effects of UVB radiation have not been reported before in spider mites. The residual effects indicated divergence in a key factor for phytoseiid mites to survive UVB radiation. In contrast, the egg hatchability and the survival rate of hatched larvae in photoreactivation experiments improved to 31.7% and 71.1% in N. californicus reared on P. citri, respectively. Many plant-dwelling mites avoid solar UVB radiation by remaining on lower leaf surfaces of host plants (Ohtsuka and Osakabe 2009; Sudo and Osakabe 2011, 2015). In contrast, P. citri is more tolerant to UVB radiation than T. urticae and resides not only on the lower leaf surfaces but also on upper leaf surfaces (Fukaya et al. 2013). P. citri possesses astaxanthin as a major pigment (Metcalf and Newell 1962, Atarashi et al. 2017), which is a prominent antioxidant among the carotenoids (Miki 1991, Kobayashi and Sakamoto 1999). Atarashi et al. (2017) showed that astaxanthin reduced the accumulation of lipid peroxide (LPO) caused by UVB- and high-temperature-generated reactive oxygen species (ROS) in P. citri. Thermal stress enhances ROS generation and LPO accumulation due to a deficiency in enzymatic antioxidant systems (catalase, superoxide dismutase, peroxidase, and glutathione S-transferase) in a phytoseiid mite Neoseiulus cucumeris (Oudemans) (Acari: Phytoseiidae), with the result that the mite died (Zhang et al. 2014). A similar deficiency in enzymatic antioxidant systems was also detected in P. citri, so that the presence of a non-enzymatic defense system was suggested (Yang et al. 2010). T. urticae does not accumulate astaxanthin in the summer form, whereas they accumulate keto-carotenoids, including astaxanthin, in the diapausing winter form (Veerman 1974, Kawaguchi et al. 2016) and increase UVB tolerance (Suzuki et al. 2009). This indicates that astaxanthin in P. citri was possibly responsible for the improvement in the hatchability of eggs and survival of hatched larvae of N. californicus. Variation in the effects of temperature on the biological impact of intermittent UVB irradiation between spider mites and phytoseiid mites may be valuable to develop appropriate irradiation conditions for an IPM strategy. Major biological damage suffered from UVB radiation is product of DNA lesions, such as cyclobutane pyrimidine dimers and pyrimidine (6–4) pyrimidone photoproducts and the generation of ROS, such as singlet oxygen (Sinha and Häder 2002). Although the impact of ROS is not necessarily lethal in spider mites (Atarashi et al. 2017), a non-enzymatic antioxidant system may be more significant in surviving UVB radiation in phytoseiid mites and their UVB tolerance may be improved by food advantageously together with the practical use of UVB lamps. The serious development of acaricide resistance in spider mites causes failure of chemical control. The combined use of UVB lamps and light reflection sheets is a novel physical control method that enables to control spider mite and powdery mildew simultaneously in strawberry greenhouse and likely to be effective also on acaricide resistant mites. However, leaf sunscald in winter by UVB irradiation is a problem for producers, and many are concerned about the mal-effects on phytoseiid mites by the UVB radiation. Our findings indicate that it will be possible to reduce UVB dose while maintaining the control effects on spider mites in winter and to use phytoseiid mites concurrently with UVB irradiation, contributing IPM strategies in strawberry greenhouse. Supplementary Material Supplementary data are available at Environmental Entomology online. Acknowledgments This work was supported by Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), ‘Technologies for creating next-generation agriculture, forestry and fisheries’ (funding agency: Bio-oriented Technology Research Advancement Institution, NARO) and JSPS KAKENHI (a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan) grant number 26292029. References Cited Atarashi, M., Manabe Y., Kishimoto H., Sugawara T., and Osakabe M.. 2017. Antioxidant protection by astaxanthin in the citrus red mite (Acari: Tetranychidae). 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Environmental EntomologyOxford University Press

Published: Feb 1, 2018

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