Aged virgin adults respond to extreme heat events with phenotypic plasticity in an invasive species, Drosophila suzukii

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Please cite this article as: Xue, Q., Ma, C-S., Aged virgin adults respond to extreme heat events with phenotypic plasticity in an invasive species, Drosophila suzukii, Journal of Insect Physiology (2020), doi: https://doi.org/ 10.1016/j.jinsphys.2020.104016

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Aged virgin adults respond to extreme heat events with phenotypic plasticity in an invasive species, Drosophila suzukii Qi Xue and Chun-Sen Ma* Climate Change Biology Research Group, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No 2, Yuanmingyuan West Road, Haidian District, Beijing 100193, PR China

* Correspondence author: Chun-Sen Ma (E-mail: [email protected], Tel. & Fax: +86 10 62811430) First author: Qi Xue (E-mail: [email protected], Tel. & Fax: +86 10 62811430)

Abstract Climate warming has increased the frequency of extreme heat events. Alien species usually invade new areas with a low-density population and often have limited mating opportunities due to the unsynchronized emergence of adults. Early-emerging virgin adults often have to wait to mate with later-emerging partners at the cost of aging, which reduces thermal tolerance. To understand the adaptive strategies of virgin males/females versus those of mated males/females in response to heat stress during aging, we conducted a fully factorial experiment to test the basal and plastic heat tolerance (CTmax, critical thermal maximum) of males and females with different mating statuses (virgin and mated) at different ages (5, 10, and 15 days after eclosion) after different acclimation regimes (null, rapid and developmental heat acclimation) in a well-known invasive species, Drosophila suzukii. We found that mating could change the heat tolerance of adults during aging. Mated females had higher basal heat tolerance than virgin females, while mated males had lower tolerance than virgin males. Mating could generally decrease the acclimation capacity (i.e., plasticity of heat tolerance) during aging. Aged virgin adults had a much higher acclimation capacity than aged mated adults. Our findings suggest that phenotypic plasticity of heat tolerance may be a main strategy used by virgin adults to cope with heat events. The phenotypic plasticity of thermal tolerance could increase the invasion success of alien species in new areas by allowing them to rapid respond to local temperature changes.

Keywords: Drosophila suzukii; Heat tolerance; Plasticity; Aging; Mating status

1. Introduction Climate warming increases the frequency and success of invasion events because it can bring about the expansion of species distributions into high-latitude or -altitude regions, reducing the competitive advantages of native species over invasive species (Dukes and Monkeys, 1999; Walther et al., 2009; Diez et al., 2012). Overwintering may be a key factor influencing the population establishment of invasive species in new areas (Leather et al., 1995; Mcdonald et al., 2000). However, population increases (Ma et al., 2015a, b) and successful expansion (Battisti et al., 2006; Pateman et al., 2012) could be strongly influenced by summer temperatures. Climate warming has increased the frequency and intensity of extreme heat events (Rahmstorf and Coumou, 2011; IPCC, 2014), which may greatly reduce the thermal safety margin of organisms (Deutsch, 2008; Paaijmans et al., 2013). Thus, the ability to buffer extreme high temperatures in summer becomes critical for successful invasion. Organisms have evolved adaptive mechanisms to buffer heat stress (Hoffmann et al., 2003; Xue et al., 2019). Phenotypic plasticity in heat tolerance improves fitness through rapid and flexible responses to temperature changes (Chown and Terblanche, 2006; Sgrò et al., 2016) without changing gene sequences/frequencies (Schlichting and Pigliucci, 1993; Schlichting and Wund, 2014). This plasticity is beneficial for invasive species to survive under extreme temperature conditions by increasing their upper thermal limits (Angilletta, 2009; Colinet and Hoffmann, 2012; Sgrò et al., 2016; Seebacher et al., 2015), which may increase their invasion success (Chown et al., 2007; Nyamukondiwa et al., 2010; Shearer et al., 2016). Therefore, a better understanding of heat tolerance and its plasticity is of great importance in the prediction of invasion risk under climate warming. Successful copulation and reproduction are essential for alien species to establish and increase

populations in new areas (Liebhold and Tobin, 2008). Adults differing in mating status may have different energy allocation strategies (Trivers, 1972; Alexander and Borgia, 1979). Females and males play different roles in reproduction. Successfully copulated females allocate more energy to reproduction, while mated males are not important in reproduction. However, virgin females or males might spend their time and energy finding suitable heterosexual individuals with which to copulate, especially when the initial population remains at a low density at the beginning of colonization and the males and females do not emerge simultaneously (Trivers, 1972; Alexander and Borgia, 1979; Slansky, 1980; Simmons et al., 1992). Thus, early-emerging virgin males or females must pay the cost of aging while waiting for copulation. Importantly, aging can significantly reduce basal thermal tolerance (Bowler, 1967; Davison, 1969; Bowler and Terblanche, 2008; Colinet et al., 2013; Chidawanyika et al., 2017) and plastic heat tolerance (Sørensen and Loeschcke, 2002; Jensen et al., 2007; Pappas et al., 2007). If extreme heat events occur during adult aging, two questions emerge: 1) Do virgin adults respond to extreme temperatures differently from mated adults during aging, and what is the potential function of such differences? 2) Do mated/virgin females respond to extreme high temperatures differently than mated/virgin males during aging, and what are the potential biological functions? Based on our literature survey, the impact of mating on heat tolerance during aging has not been studied in detail, although mating can weaken the cold tolerance of adults in Ophraella communa (Zhao et al., 2019) and Harmonia axyridis (Facon et al., 2017). Mating could benefit females in toleration of starvation and desiccation. Mated females are more tolerant than virgin females to starvation stress in Drosophila melanogaster (Service, 1989; Rush et al., 2007; Goenaga et al., 2012), and desiccation in two desert Drosophila (Drosophila mojavensis and Drosophila

arizonae) (Knowles et al., 2004; 2005). Previous studies have mainly focused on the impact of mating on basal stress tolerance, while the impact of mating on acclimation capacity (i.e., phenotypic plasticity of stress tolerance) has not been directly investigated clearly. We selected spotted wing drosophila (SWD), Drosophila suzukii Matsumura (Diptera: Drosophilidae), a widely investigated invasive species in recent years (Asplen et al., 2015), as our model system. SWD is native to Asia and was first described by Matsumara in 1931 (Bächli, 2015). It has thus far successfully colonized the Americas and Europe (Kanzawa, 1939; Cini et al., 2012; Asplen et al., 2015). In contrast to Drosophila melanogaster, SWD has caused severe economic damage in invaded countries because it has a serrated ovipositor and exclusively lays eggs inside many preharvest or ripe soft-skinned fruits, such as berries, cherries, and grapes (Walsh et al., 2011; Karageorgi et al., 2017). Thus, potential invasion risk assessment of SWD based on basal and plastic thermal tolerance has become important (Valladares et al., 2014; Langille et al., 2017; dos Santos et al., 2017). The cold tolerance of SWD adults in the overwintering stage has been intensively investigated (Enriquez and Colinet, 2017; Ryan et al., 2016; Jakobs et al., 2015; Wallingford and Loeb, 2016), and used to assess the invasion risk (Jakobs et al., 2015; Kaçar et al., 2015; Plantamp et al., 2016; Tonina et al., 2016). However, the heat tolerance of SWD has not been largely investigated. A better understanding of heat tolerance and its phenotypic plasticity during aging will be helpful in invasion risk assessment under climate change. Here, we measured the basal and plastic heat tolerance (CTmax) of SWD male and female adults with different mating statuses (mated and virgin) and ages (5, 10, and 15 days). Furthermore, we calculated the acclimation capacity i.e., phenotypic plasticity of heat tolerance, based on the method of Kellett et al. (2005). We aimed to clarify whether virgin males and females waiting for

copulation have different stress tolerance strategies than successfully mated adults during aging and the biological significance of such strategies. Our results indicate that mating could change the heat tolerance of adults during aging. Mated females had higher basal heat tolerance than virgin females, while mated males had lower tolerance than virgin males. Mating could also generally decrease acclimation capacity. Aged virgin adults had a much stronger acclimation capacity than aged mated adults. Phenotypic plasticity may be a beneficial approach for virgin adults to adapt to extreme temperature conditions during aging, which would help adults of alien species increase the probability of successful copulation and reproduction.

2. Material and methods 2.1. Fly culture The SWD used in the experiments were collected from a stock-reared population that was established in 2017 and introduced from the MoA-CABI Joint Laboratory for Bio-Safety (Institute of Plant Protection, Chinese Academy of Agricultural Sciences). The flies were originally collected in Beijing, China (Haidian district), from infested cherries (Cerasus pseudocerasus) in 2014. The laboratory population was maintained at approximately 400 individuals in two screen cages (30×25×30 cm) and supplied with artificially wounded table grapes (‘Kyoho’ grapes, Vitis vinifera) in a Petri dish (9 cm diameter), which were purchased from markets; the artificial wounds, approximately 1 cm in size, were produced using a scalpel after fully cleaning the grapes with water. The rearing cages were placed in a climatic chamber (RXZ-380B; Jiangnan Ltd., China) at 23± 0.5°C and 70±10% RH and under a photoperiod of 16 L: 8 D. The grapes were renewed every 2 days to provide larvae with sufficient food and development space. In addition, a cotton ball soaked with a 5% honey solution was provided for adult feeding.

2.2. Experimental design and treatments To evaluate the effects of mating status (mated or virgin), adult age (5, 10, or 15 days), sex (female or male) and acclimation pattern (null, rapid or developmental acclimation) on the heat tolerance of SWD adults, we used a fully factorial experimental design involving 3×2×2×3 treatments. We measured the CTmax of 20 females and 20 males in each treatment. We tested the CTmax of flies with null acclimation, i.e., the basal heat tolerance by rearing all stages of flies at a constant 23°C and 70% RH in climatic chambers without heat stress before the measurements. We measured the CTmax of rapidly acclimated flies (exposure to 30°C for 2 h and then recovery at 23°C for 1 h before CTmax measurement) and developmentally acclimated flies (exposing individuals in the whole egg, larval and pupal stages to 28°C and then shifting to 23°C after eclosion before CTmax measurement). All thermal acclimation treatments were conducted in climate chambers, and the temperature and relative humidity inside were recorded with HOBO data loggers (U23-001; Onset Ltd., Bourne, MA, USA).

2.3. Experimental manipulation To obtain flies for the experiments, we collected eggs by placing artificially wounded grapes in Petri dishes (diameter 9 cm) in the stock rearing cages of the SWD population. The grapes for egg collection were renewed every 2 days to maintain favorability for oviposition by adults and to avoid overcrowding the environment of larvae during development. To study the effect of adult age on basal and acclimated heat tolerance, the newly emerged adults (within 6 h) in the different treatments were reared for 5, 10, 15 days at 23°C and 70% RH and under 16 L:8 D before measuring CTmax. To assess the differences in the basal and acclimated heat tolerance of adults with different mating statuses, we obtained virgin and mated adult flies after emergence through the following

manipulations. To obtain virgin adults, we individually reared adults after emergence using glass tubes (diameter 1.5 cm, length 10 cm) supplied with a cotton ball containing a 5% honey/water solution. Correspondingly, one male and one female were paired and reared in centrifuge tubes (capacity 50 ml). The bottoms of these tubes were removed and covered with gauze. The lids of the tubes were filled with artificial medium made from a formula (200 ml grape juice + 3 g agar + 30 ml distilled water) to provide sites for the females to lay eggs. The lid contents were renewed every 3 days to assure wetness and no microbial degradation. Furthermore, we carefully checked for the appearance of any eggs within the medium in these lids under a microscope and considered the adults in tubes and lids with eggs as successfully mated adults (the number of matings was not considered). These mated adults also refer to reproductive adults. The critical thermal maximum (CTmax) was used to indicate heat tolerance and acclimated heat tolerance. The detailed method was described in Zhao et al. (2017). We used the same protocol but modified the ramping rate (see below). First, the tested adults were placed individually inside 24-well cell culture plates. To minimize potential margin effects on the tested adults, we only placed 8 adults individually into the 8 wells in the central part of the plate. Then, the plate was placed vertically in the arena, a double-layer glass container (diameter 30 cm, height 20 cm), in which temperature could be controlled by heating the interlayer with a glycol bath (Ministat 230-cc-NR; Huber Ltd., Germany, accuracy ± 0.01°C). The temperature within the container was monitored by a thermosensor linked to the glycol bath. After maintaining the adults at 23°C for 15 min, the temperature within the container was gradually increased at a rate of 0.1°C min−1 until all adults died (less than 38°C), and the action of flies was recorded by DV (Panasonic HDC-HS700) during the process of heating. The temperature at which an adult fly lost the ability to move and showed

spasms was recorded as its CTmax by watching the videos.

2.4. Statistical analysis We used generalized linear models (GLMs) with normally distributed errors to determine the effects of three main factors (mating status, sex and adult age) and their interactions on basal, rapid and developmental acclimation CTmax with the ‘glm’ function and ‘car’ package in R. Multiple comparisons of basal heat tolerance between mated and virgin adults were analyzed using the Tukey post hoc test with the ‘glht’ function in the ‘multcomp’ package. To compare the rate of decline in basal and plastic heat tolerance with aging among the different treatments, we performed pairwise comparisons of slopes among the different treatments using ANCOVA with the covariate of adult age (SPSS v. 13.0). Finally, according to the definition of acclimation capacity (acclimation capacity = acclimated CTmax − basal CTmax) (Kellett et al., 2005), we used an independent-samples t-test to estimate the acclimation capacity and the associated s.e.m. in SPSS (Zhao et al., 2017).

3. Results 3.1. Mating status determined the age-dependent basal heat tolerance of adults In terms of the impacts of the three factors on the basal heat tolerance (CTmax) of adult flies, the three-way interaction among age, sex and mating status was not significant, but the two-way interactions between mating status and age or sex were highly significant (Table 1). Mating status (χ2 = 16.05, p < 0.001) rather than sex (χ2 = 4.33, p = 0.115) determined the age-dependent change rate of CTmax. The CTmax of mated females declined more slowly than that of virgin females during aging (Fd.f. = 9.271,118, p = 0.003). The impact of mating status on the decline rate of the CTmax of males during aging was similar to that of females (Fd.f. = 4.081,109, p = 0.046) (see Fig. 1, Table 2). In addition, mating increased heat tolerance in 15-day-old females (t = 3.890, df = 37, p =

0.0004) and decreased heat tolerance in 10-day-old males (t = -2.45, df = 35, p = 0.019) (see Fig. 2).

3.2. Mating status affected the age-dependent acclimated heat tolerance of adults In terms of the impacts of adult age, sex and mating status on the rapid acclimation heat tolerance (CTmax) of adult flies, the three-way interaction among the three factors and the two-way interaction between age and sex were significant (Table 1). Mating status did not (χ2 = 2.64, p = 0.267) but sex did affect (χ2 = 7.08, p = 0.029) the age-dependent rapid acclimation heat tolerance. Among the virgin adults, the rapid acclimation CTmax of females decreased faster than that of males (Fd.f. = 5.931,111, p = 0.016), while among the mated adults, the CTmax of the different sexes did not significantly differ (Fd.f. = 0.361,116, p = 0.549) (see Table 2; Fig. 1). Overall, rapid heat acclimation enhanced CTmax by an approximate maximum of 1.5°C (see Fig. 3). Young virgin female adults always had higher acclimation capacity than young mated females, and the plasticity of virgin females increased as they aged, while that of mated females decreased as they aged (Fig. 3). Aged virgin male adults had a higher acclimation capacity than aged mated males (Fig. 3). The highest plasticity was found at the age of 15 days for the virgin adults, while the opposite result was observed for the mated adults (Fig.3). In terms of the impacts of the three factors of adult age, sex and mating status on developmentally acclimated CTmax, the three-way interaction of the main factors was not significant, but the two-way interaction between mating status and age or sex was significant (Table 1). Mating status (χ2 = 11.73, p = 0.003) rather than sex (χ2 = 3.76, p = 0.153) affected the agedependent developmentally acclimated CTmax. The CTmax of the mated males declined faster than that of the virgin males (Fd.f. = 5.491,91, p = 0.021), while the declining slopes of CTmax between

the different mating statuses of females did not significantly differ (Fd.f. = 0.021,113, p = 0.891) (see Table 2; Fig. 1). Developmental heat acclimation generally increased CTmax by an approximate maximum of 1.5°C (Fig. 3). Virgin male and female adults always had a higher acclimation capacity than mated male and female adults (Fig. 3). Aged virgin males (15 days) had a much higher acclimation capacity than aged mated males. The plasticity of males was larger than that of females despite the mating status of adults.

4. Discussion The ability to buffer extreme high temperatures in summer has become critical for successful invasion. The phenotypic plasticity of heat tolerance may contribute to an increase in the survival of invasive species at extreme temperatures and dispersal to more stressful areas (Chown et al., 2007; Nyamukondiwa et al., 2010; Shearer et al., 2016). Early-emerging virgin males or females must pay the cost of aging when waiting for copulation, which can reduce both basal (Bowler, 1967; Davison, 1969; Bowler and Terblanche, 2008; Colinet et al., 2013; Chidawanyika et al., 2017) and plastic heat tolerance (Sørensen and Loeschcke, 2002; Jensen et al., 2007; Pappas et al., 2007). To understand whether virgin males/females versus mated males/females have different heat stress adaptation strategies during aging, we used D. suzukii as our model system to test the basal and induced heat tolerance of males and females with different mating statuses at different ages. The results showed that mating could significantly change the heat tolerance of adults during aging. Males and females responded to high temperatures during aging differently. Overall, mated females had higher basal heat tolerance than virgin females, while mated males had lower tolerance than virgin males. In addition, mating could also decrease the acclimation capacity, and aged virgin adults had much higher acclimation capacity than aged mated adults. Virgin adults generally showed

enhanced plasticity in terms of adapting to extremely high temperatures during aging (especially 15-day-old virgins), which would help alien species increase the probability of subsequent successful copulation and reproduction.

4.1. Mating could alter the basal heat tolerance of adults We found that mated females have higher basal heat tolerance than virgin females, while mated males have lower basal heat tolerance than virgin males. To the best of our knowledge, this phenomenon in regard to heat tolerance has not yet been reported. However, similar performance was found in desiccation tolerance (Knowles et al., 2004; 2005) and starvation tolerance in Drosophila melanogaster (Service, 1989; Rush et al., 2007; Goenaga et al., 2012). The starvation and desiccation tolerance of successfully mated females in D. melanogaster were higher than those in virgin females, whereas these stress tolerances in mated males were lower than those in virgin males. The reason for this phenomenon may be related to changes in the physiological conditions of adults after successful mating. During mating, males may transfer many nuptial gifts to females, such as water, seminal fluid proteins (SFPs) and sex peptides (SPs) (Edvardsson, 2007; Gwynne, 2008; Goenaga et al., 2012). The nuptial gifts provide females with water and energy-containing substances and facilitate female feeding through an intrinsic mechanism, which allows females to gain more water and energy resources in turn (Carvalho et al., 2006; Avila et al., 2011). In contrast, for males, mating causes the loss of water and proteins from their bodies, which may lead to the allocation of the limited water and energy to tolerate other adverse environmental stresses. These results suggest that mated adults may have evolved some adaptive strategies to cope with environmental stresses to produce more offspring and maintain the population. Multiple mating impacts cold tolerance, with the cold tolerance of reproductive females (laying

eggs) being significantly lower than that of multiple-mated females (mated at least once but having laid no eggs) and virgin adults of Harmonia axyridis (Facon et al., 2017). Multiple mating behavior has been demonstrated both in Drosophila melanogaster (Markow, 2002) and Drosophila suzukii (Revadi et al., 2015, Krüger et al., 2018). However, there is no information about the impacts of multiple mating on heat tolerance. We only focus on two status (virgin versus adults normally mated and laid eggs) without considering impacts of multiple mating on heat tolerance in the present study, and it needs to be conducted in further investigations.

4.2. Mating could alter the plasticity of heat tolerance in adults We calculated the acclimation capacity in accordance with previous studies (see Kellett et al., 2005; Zhao et al., 2017). We newly found that mating could alter the acclimation capacity, i.e., the plasticity of heat tolerance. The acclimation capacity of mated adults decreased with aging, whereas that of virgin adults increased with aging (see 15-day-old adults). We found that the acclimation CTmax of mated adults declined faster than that of virgin adults during aging, while the basal CTmax of mated adults declined more slowly than that of virgin adults. This variation in acclimation CTmax and basal CTmax resulted in different acclimation capacities of mated and virgin adults during aging. The main difference between mated and virgin adults is the output of reproduction, which consumes much energy and nutrients in adults (Harshman and Zera, 2007). Furthermore, heat tolerance also requires energy. The expression of heat shock proteins (Hsps) may be involved in regulating the plasticity of heat tolerance (Hu et al., 2014) because Hsps are beneficial for adults to improve their heat tolerance (Hoffmann et al., 2003; Zhang et al., 2016). The synthesis of Hsps also requires much energy (Mallouk et al., 1999). However, the expression of Hsps could be limited by reproduction and aging. For example, in Drosophila melanogaster, Hsp70 expression is

downregulated with aging (Sørensen and Loeschcke, 2002) and reproduction (Silbermann and Tatar, 2000). Therefore, the difference between mated and virgin adults in acclimation capacity might be related to the difference in energy allocation to reproduction and heat tolerance between mated and virgin adults. Mated females might allocate more energy to reproduction, while virgin males and females allocate more energy to heat tolerance. The energy available to mated adults for the acclimation capacity of heat tolerance might be less than that available to virgin adults.

4.3. The ecological function of different heat adaptive approaches in virgin or mated adults Organisms may have two fundamental approaches to cope with extreme heat stress: 1) having higher basal heat tolerance (evolutionary and genetic adaptation) and 2) inducing stronger heat acclimation capacity (plastic adaptation) (Esperk et al., 2016; Sgrò et al., 2016). Interestingly, we found that aged virgin adults (15 days) had significantly higher absolute acclimation capacity than mated adults of the same age, but the basal heat tolerance of aged virgin female adults (15 days) was lower than that of mated female adults. Virgin adults may cope with extreme heat stress through strengthened acclimation capacity; this approach allows them to rapidly adapt to extreme heat stress and consume a lower amount of energy than that under the other approach (i.e., having higher basal heat tolerance). Plastic heat tolerance is conditionally induced by acclimation temperature and rapidly responds to heat stress (Chown and Terblanche, 2006; Sgrò et al., 2016) and conserves energy invested in stress tolerance (Davies et al., 2015), likely because this approach can avoid energy consumption for maintaining higher basal heat tolerance through the continued synthesis of Hsps (Rinehart et al., 2006). Virgin adults might need to allocate more energy to subsequent copulation and reproduction (Arnqvist and Rowe, 2005), and the response to extreme heat stress by

inducing stronger heat acclimation capacity might be beneficial for their population. Among mated females, the laying of more eggs is important for maintaining the population (Arnqvist and Rowe, 2005), while reproduction can consume lots of energy (Harshman and Zera, 2007). Thereby, the adaptive approach to heat stress that consumes less energy may benefit their reproduction. Mating can improve the basal heat tolerance of females, thus to some extent, mated females may don’t need to consume a lot of energy for improvement of heat tolerance, which may be beneficial for them to allocate more energy to reproduction. Together, the different heat adaptive strategies adopted by mated and virgin adults may be an attempt to allocate more energy to reproduction, which will greatly contribute to population colonization. D. suzukii has invaded many countries (Asplen et al., 2015). This species is reported to have evolved in mountain regions and hence is well adapted to cool climates (Ometto et al., 2013). However, we found that the basal heat tolerance of D. suzukii adults approximately was 31-35°C and the maximum plasticity of heat tolerance approximately was 1.5°C. When D. suzukii are introduced into a new environment, the plasticity may greatly improve their survival over short timescales. Thus, our results may be helpful in interpreting the rapid expansion of D. suzukii in the Southern Hemisphere, as models predict that D. suzukii can potentially be distributed in the western and eastern regions of the South African and Australian subtropics (dos Santos et al., 2017). In addition, the winter adult morphotype (Shearer et al., 2016) might be accidentally transported to the summer environment. There are differences in physiological conditions between mated and unmated winter morphs of D. suzukii because sperm are found in the ovaries of mated winter morph females (Grassi et al., 2017). The differences in physiological conditions may have different effects on thermal tolerance. For the winter morph of the ladybird Harmonia axyridis, the cold survival

capacity of mated females is lower than that of virgin females (Facon et al., 2017). However, we do not know the exact difference in thermal tolerance between mated and unmated winter morph individuals, and more investigations are required in the future.

4.4. Conclusions Alien species usually invade new areas with a low-density population and often have limited mating opportunities due to the unsynchronized emergence of adults at low density. Early-emerging virgin males or females must pay the cost of aging while waiting for copulation, and they may face serious heat stress because extreme high-temperature events resulting from climate warming often occur in summer and have important impacts on organisms (Ma et al., 2015a, b). We found that virgin adults of D. suzukii can compensate for a decrease in basal heat tolerance during aging by increasing the plasticity of heat tolerance, which may favor survival and the probability of successful copulation and reproduction, thereby increasing the success of invasion. Our findings may be of significance in the interpretation of the mechanisms of rapid expansion of D. suzukii to many countries. These results suggest that the high plasticity of thermal tolerance among virgin adults may be an important approach for alien species to adapt to local climatic conditions and successfully colonize with a low-density initial population when introduced into a novel environment. Thus, for the purpose of the accurate assessment of invasion risk and the effective management of alien species, we should consider not only mated adults because they can produce offspring but also virgin adults because they have great potential in survival and subsequent copulation and reproduction in adverse environments. Models of population dynamics and the risk assessment of invasive species can be developed based on the demographics of populations that are collected under simulated environmental conditions (Tanga et al., 2018; Yee et al., 2017). We need to consider the mating

status of adults in these models to improve their prediction accuracy, especially for the low-density initial colonization.

Acknowledgements We thank Xue-Jing Wang and Liang Zhu for constructive discussion with this manuscript. We thank MoA-CABI Joint Laboratory for Bio-Safety (Beijing) for providing population source of Drosophila suzukii.

Author statements The authors declare no competing or financial interests. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Author contributions C.S.M and Q.X conceived and designed the experiments. Q.X performed the experiments. Q.X and C.S.M analyzed the data, and then wrote the manuscript. All authors reviewed and approved the manuscript.

Funding This work was mainly financially supported by the earmarked fund of China Agriculture Research System (CARS-29-bc-4) and National Key R&D Program of China (2018YFD0201300).

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Table 1. Effects of age, mating status and sex on basal, rapid and developmental acclimation heat tolerance (CTmax). Values indicate chi-square statistics of GLMs and corresponding p values. Significant values are indicated in bold. Basal CTmax

Rapid acclimation

Developmental

CTmax

acclimation CTmax

　

Source

df

χ2

p

χ2

p

χ2

p

Age

2

29.41

<0.001

36.26

<0.001

30.54

<0.001

Sex

1

75.53

<0.001

77.36

<0.001

12.78

<0.001

Mating status

1

1.93

0.165

6.51

0.011

27.08

<0.001

Age × Sex

2

4.33

0.115

7.08

0.029

3.76

0.153

Age × Mating status

2

16.05

<0.001

2.64

0.267

11.73

0.003

Sex × Mating status

1

16.45

<0.001

0.02

0.887

14.54

<0.001

Age × Sex × Mating status

2

0.53

0.767

6.47

0.039

3.15

0.207

Table 2. Effects of mating status and sex on slopes of linear regressions of age-dependent basal, rapid acclimation and developmental acclimation heat tolerance (CTmax).

Basal heat tolerance

Rapid acclimation

Developmental

response

acclimation response

Fd.f.

p

Fd.f.

p

Fd.f.

p

virgin: F vs M

3.111,112

0.081

5.931,111

0.016

0.421,103

0.517

mated: F vs M

1.271,115

0.262

0.361,116

0.549

3.061,101

0.083

F: mated vs virgin

9.271,118

0.003

0.031,117

0.867

0.021,113

0.891

M: mated vs virgin

4.081,109

0.046

2.411,110

0.123

5.491,91

0.021

Curve comparisons

F represents females, M represents males

Fig. 1. Effects of adult age, mating status and sex on basal, rapid acclimation and developmental acclimation heat tolerance (CTmax) of Drosophila suzukii. Different colors represent different

treatments. Panels indicate the sex of the flies. Points represent individuals and were jittered to avoid overlap. The solid lines represent basal tolerance, and the dashed lines represent rapid or developmental tolerance. The shaded areas represent the 95% confidence intervals (CIs) of the fit performed in the statistical analysis of the data.

Fig. 2. Effects of mating status and adult age on heat tolerance of female and male adults. Bars represent the mean heat tolerance (CTmax) ± s.e.m. Stars above the boxes represent a significant difference in heat tolerance (CTmax) between mated and virgin adults. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3. Absolute acclimation capacity (mean ± s.e.m) under different treatments of age, mating status and acclimation form, including rapid acclimation (RA) and developmental acclimation (DA).

Ethical approved and Declaration of competing interests All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The authors declare that they have no conflict of interest.

Author contributions statement C.S.M and Q.X conceived and designed the experiments. Q.X performed the experiments. Q.X and C.S.M analyzed the data, and then wrote the manuscript. All authors reviewed and approved the manuscript.

Highlights: 

Mating may alter heat tolerance, its plasticity and their decline rate during aging.



Mated and virgin adults may take different strategies to cope with heat stress.



Rise of basal heat tolerance may benefit mated females to buffer heat stress.



Plasticity of heat tolerance may benefit virgin adults to buffer heat stress.



Mating status of adults should be consider in invasive risk assessment.