Seasonality but not photoperiodism affects 2 susceptibility of the two-spotted spider mite, 3 Tetranychus urticae Koch (Acari: Tetranychidae) to pesticides

18 Understanding how endogenous and exogenous factors such as annual seasonal rhythm and photoperiodism affect the toxicity of pesticides can help design integrated pest management strategies. The two-spotted spider mite 20 Tetranychus urticae Koch (Acari: Tetranychidae), a worldwide phytophagous pest species distributed in areas with 21 different time zones, is a good model to explore how the photoperiodism and seasonality affect the pesticide toxicity. 22 In this study, we conducted a laboratory experiment from March 2017 to November 2018 where spider mites were 23 reared at three photoperiod regimes in environmentally controlled incubators. The toxicities of two acaricides, 24 propargite and diafenthiruon, were measured on T. urticae every month. To determine potential mechanisms 25 underlying the changes in the acaricide toxicity, we measured body size and total GSTs activity with the toxicity 26 measurements in 2018. Our results showed that the toxicities of the two acaricides were not significantly different 27 among the three photoperiod regimes. However, both had a consistent trend along the season which increased in the 28 spring and summer and decreased in the winter in all photoperiod regimes for two consecutive years, suggesting 29 seasonality be an endogenous factor affecting the susceptibility of the spider mites to pesticide. Pearson’s correlation 30 analyses showed only the body size had a weak negative correlation with the acaricide toxicities, suggesting a minor 31 contribution to the higher toxicity from the smaller size of T. urticae . Our study is the first to show that seasonality 32 can have an endogenous effect on the pesticide toxicity, and the results can inform practical insights into the pest 33 management strategies.

Understanding the efficacy of chemical applications to pest species under different rhythmicity such as 36 photoperiodism and seasonality can lead to better pest management strategies [1,2]

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The two-spotted spider mite, T. urticae, is a worldwide phytophagous pest and has caused 69 significant yield loss in multiple crops, including fruits, cotton, vegetables, and ornamentals [16,18].  58.6, P < 0.0001, Fig. 1 d). However, both the photoperiod and the interaction terms did not have any 165 effects on the toxicities for both acaricides (Fig 1 a-d). In 2017, the susceptibility of T. urticae to both 166 acaricides had same levels from March to September, started decreasing from October, and reached their 167 minimum on December (Fig 1). For example, the LC 50 of diafenthiruon on T. urticae at Photoperiod 168 12L:12D increased from 7.5 (95%CI: 5.0 -11.5) mg/L on March to 17.1 (95%CI: 14.5 -20.2) mg/L on 169 December (Fig 1 a), and the LC 50 of propargite at Photoperiod 12L:12D increased from 190.1 (95%CI:  (Fig 1 b). 185

Correlation between body size and acaricide toxicity
186 The body size of T. urticae females was measured every month from January to November 2018 to 187 investigate potential correlation with acaricide toxicities. Our results suggested that the body size of T. 189 and the interaction term (F (2, 1616) = 8.9, P < 0.0001) (Fig 2). The post hoc analyses using Tukey's HSD 190 method suggested that T. urticae reared on 10L:14D has the largest body size, but the body size of the . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted March 9, 2022. ; https://doi.org/10.1101/2022.03.09.483610 doi: bioRxiv preprint mites reared on 12L:12D and 18L:6D were not significantly different ( Table S1). The body size of T.

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urticae reared on three photoperiod regimes varied along the time, but there was not a pattern being 193 observed (Fig 2, Table S1).  Table 1), suggesting the smaller body size may be more sensitive to acaricides.  The coefficients r with 95% confidence interval (95% CI) between the measured variables (including the body size 211 and GSTs activity) and the toxicities of two commonly used acaricides (including diafenthiruon and propargite) 212 were estimated by Pearson's correlation analyses.

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Seasonal variation of GST activities in T. urticae has no correlation with acaricide toxicity

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The total GSTs activity of T. urticae was measured every month from January to November 2018. A 215 linear mixed model was applied to determine the effects of time, photoperiod, and their interaction on the 216 GST activities. The results suggested that the total GSTs activity of T. urticae was affected by the time

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In this study, we investigated the effects of seasonality and photoperiodism on the toxicities of two 236 acaricides to control the two-spotted spider mite, T. urticae. Our results showed that acaricide toxicities 237 were not significantly different among the three photoperiods (10L:14D, 12L:12D and 18L:6D).

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However, toxicities changed consistently across the seasons even though other abiotic factors including 239 photoperiod, temperature, and relative humidity were controlled. Toxicities increased in spring and 240 summer (from March to September) and decreased in the winter (from October to February in the next

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year) in all photoperiod regimes. We measured the body size and total GSTs activity every month from 242 January to November in 2018 to determine their correlation to the toxicities. Even though the body size 243 and total GSTs activity were found to be affected by both photoperiodism and seasonality, there was 244 only a weak negative correlation between the body size and the toxicities, and no correlation between 245 the total GSTs activity and the toxicities.

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Our results showed that the photoperiodism affects physiology of T. urticae including the body 247 size and total GSTs activity, but not the pesticide toxicity. This is consistent with our previous study

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In conclusion, we used a worldwide agricultural pest, T. urticae, as a model to investigate how 296 photoperiodism and seasonality affect pesticide toxicity. The results showed that seasonality but not 297 photoperiodism affects pesticide toxicity of propargite and diafenthiruon on T. urticae. The toxicities of . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted March 9, 2022. ; https://doi.org/10.1101/2022.03.09.483610 doi: bioRxiv preprint both pesticides were found not significantly different among the three photoperiod regimes. However,

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the toxicities were found cyclically changing over the season which increased in the summer and 300 decreased in the winter. We found photoperiodism and seasonality had complex effects on the body 301 size and GSTs activity, but only the body size had a weak negative correlation with the pesticide 302 toxicities. Our study is the first to show that seasonality has an endogenous effect on the pesticide 303 toxicity, which is important for pest control practice in the field.