Prey life stages modulate effects of predation stress on prey lifespan, development, and reproduction in mites

The non‐consumptive effects of predator‐induced stress can influence a variety of life‐history traits. Many previous studies focused only on short‐term effects such as development and reproductive rates. Recent studies have showed that long‐term predation stress (given during the whole life of the prey) and short‐term predation stress (provided during the immature stage of the prey) could generate completely opposite results: the former could decrease lifespan, whereas the later could increase lifespan. However, it is still unclear whether the advantage is because of the short duration of exposure or the early stage of life during which exposure was exerted. Thus, in this study, the prey (Tyrophagus putrescentiae) was exposed to predation stress from the predator (Neoseiulus cucumeris) during different life stages (larva, protonymph, tritonymph, first 5 d of oviposition, the full lifespan or none of the above). The results showed that the predation stress supplied during larval and protonymphal stage delayed development, reduced fecundity and prolonged lifespan of the prey, while the stress given during tritonymphal stage only reduced lifespan slightly and the stress given during the first 5 d of oviposition did not change lifespan but reduced fecundity. This study indicated that the effects of predation stress are dependent on prey life stage and the predation stress experienced in the early life stages is important to lifespan modulation.


Introduction
A considerable body of literature in the past two decades has shown significant non-consumptive effects on life history traits, such as development (e.g., Peckarsky et al., 2002;Griffis-Kyle & Ritchie, 2007;Thaler et al., 2012), and reproductive rates (e.g., Dahl & Peckarsky, 2003;Zanette et al., 2011;Li & Zhang, 2019a). Most of these studies examined the short-term effects of immature development, reproduction, and behavior (e.g., Warkentin, Correspondence: Zhi-Qiang Zhang, School of Biological Sciences, the University of Auckland, Auckland, New Zealand. Email: zhangz@landcareresearch.co.nz 1995; Abrams & Rowe, 1996;Oku et al., 2003;Choh et al., 2010;Rocha et al., 2020;Oliveira & Moraes, 2021;Majchrzak et al., 2022;Saavedra et al., 2022). Only recently have a few studies focused on the effects of predation stress on lifespan, showing that predation stress prolongs development, reduces reproductive rates or fecundity, and shortens lifespan (Li & Zhang, 2019a;. Our experiments (Li & Zhang, 2019a; showed that predation cues from as low as only one predator could last for all the lifespan of the prey. The results did not confirm the hypothesis that milder levels of predation stress may have no effect on prey lifespan or even increase it. However, one later experiment of  showed that predation stress created during the immature stage showed totally opposite results from predation stress during oviposition and post-oviposition periods. The experiment indicated that exposure to a milder level of predation stress in the form of short-term exposure during immature stages could prolong lifespan, which confirmed the hypothesis of . However, it is still unclear if the benefits of this "mild" predation stress resulted from the short duration of exposure or from the early stage of life during which exposure was exerted. Many studies, which focused on factors such as temperature and fasting, showed similar trends. Exposure to both extreme nonlethal chilling and heat decreased the lifespans of females (Søvik & Leinaas, 2003;Ayar et al., 2009;Mironidis & Savopoulou-Soultani, 2010;Wang et al., 2014;Jiao et al., 2016;Zhang et al., 2016). However, repeated exposure to heat or cold stress could generate totally opposite results of increased lifespan and heat resistance (Hercus et al., 2003;Søvik & Leinaas, 2003;Defays et al., 2011;Sarup et al., 2014;Zheng et al., 2017). Moreover, mild fasting (diluted medium for 2 d) could increase the survival to anoxia stress (Vigne et al., 2009), and repeated short-period fasting (relatively mild) could extend lifespan (Varady & Hellerstein, 2007;Colman et al., 2009Colman et al., , 2014Singh et al., 2012;Li & Zhang, 2019b;Caramoci et al., 2016). However, in most of these studies, short-term stress was provided repeatedly not just given during young age. According to Le Bourg (2007), the short-term coldness given during the young age could increase the lifespan of male flies (Drosophila melanogaster). However, it is still unknown whether the benefits generated by such mild stress were coming from the short periods or from the young age.
Thus, in this study, we exposed prey to predation stress during each immature stage as well early adult stage for only a few days by using the prey-predator model of T. putrescentiae and N. cucumeris (details see Wei & Zhang, 2019). This system made it convenient to supply predation cues as it allowed the cues to be transmitted to the prey without direct contact of the predator and the prey. This allowed the provision of predation cues either during the whole of life or only during different life stages of the prey. This study aims to test the effects of short exposure of predation stress given during different immature stages as well as early adult stage on all lifespan traits, including lifespan and fecundity.

Mite colony
In this study, we selected the storage mite T. putrescentiae (Acari: Acaridae) as the experimental prey species, and N. cucumeris (Acari: Phytoseiidae) was used as the predator. Both were used in mass production of commercialized and laboratory populations and T. putrescentiae was a well-known factitious prey of this predatory mite species (Lee et al., 2020). Both species are easy to accommodate under laboratory environment. The original colonies of both species were obtained from Bioforce Limited in South Auckland. The experimental colony of T. putrescentiae was maintained in a cylindrical transparent plastic box (15 × 10 × 7 cm), while the colony of N. cucumeris was maintained in a 1000-m transparent plastic box (15 × 10 × 7 cm), which was kept in plastic containers (35.5 × 23.5 × 12.0 cm) with water. T. putrescentiae was feed on yeast (produced by Goodman Fielder Limited, New Zealand), while N. cucumeris was feed on T. putrescentiae. Both colonies were kept in a room controlled at temperature (25 ± 2°C), humidity (80% ± 2%) and photoperiod (16 L : 8 D).

Experimental setups
Control group without predation risk The Munger cell was made by 2 pieces of identical plexiglass (25 mm wide, 38 mm long and 3 mm thick). One piece of plexiglass had a cone-shape hole (top diameter 9 mm, bottom diameter 7 mm) in the middle, the other did not. The plexiglass without the hole was clipped on top of the other piece of plexiglass, while the plexiglass with the hole had covering of a mesh material (500 grids per square inch) on the bottom. One T. putrescentiae was kept in each cell. A small drop of yeast paste was applied on the mesh material in the cell by using a fine hair-brush (size 000) to feed the storage mite (details see Wei & Zhang, 2019).

Treatment groups
The Munger cell of treatment group was made up of 2 parts: the top part was the same as the control cell, while its bottom was clipped with another identical plexiglass with a hole. The top of this second plexiglass was also covered by a piece of mesh (500 grids per square inch). Thus, the top cell was used to contain the prey, while the bottom cell was used to contain the predators. This allowed them to feel each other but without direct contact due to the screen between 2 cells (details see Wei & Zhang, 2019).

Experimental procedures
Standardization of test prey Eggs of T. putrescentiae were picked using a hair-brush (size 000) from the main colony and placed in groups of 20 into a Munger cell with a diameter of 15 mm in a plexiglass slide (40 × 40 × 3 mm). Several of these cells were prepared. Mites were fed yeast. When mites developed to adults, they were allowed to mate for a few days. Then young gravid females of similar ages were placed into new cells for 24 h to lay a new generation of eggs. The eggs laid within 24 h were used for experiments.
Experiments Eggs laid on the same day were placed into separate Munger cells and were alone in the cell until they developed to adults. A male and a female of the same treatment were paired in each Munger cell and the pairs were observed until they were dead. To measure the size, the dead mites were mounted in Hoyer's medium on a glass slide. A Nikon microscope was used to measure the length of the prodorsal plate (an index of body size). Predation pressure was applied by placing 5 adult predators into the neighboring cell (predator cues such as odor could transmit to prey cell via the mesh screen) and was given during different life stages (larva, protonymph, tritonymph, and adult) of the prey in various treatments. For every 5-7 d, food for prey and the predator mites was replaced to ensure the freshness of the food and the activity of the predators. There were 6 groups for this study: (1) the Control group: the prey did not have predation stress during their whole lifespans; (2) the L group: the prey had predation stress during the larval stage; (3) the P group: the prey was given predation stress during the protonymphal stage; (4) the T group: the predation stress was given during the tritonymphal stage of the prey; (5) the Ovi group: the prey had predation stress during the first 5 d of the oviposition period; and (6) the Full group: the prey in this group had predation stress during their whole lifespans. In each group 50 replicates were set up.

Statistics
All data were analyzed in R software (vension 4.1.1). Because development and lifespan data did not follow the normal distribution (although having the homogeneity of variance), the non-parametric 2-factor analysis method, Scheirer-Ray-Hare test was used to analyze the effects of predation stress and gender on the development time and lifespan of the mites and the interaction between the 2 factors. Although the data of the post-oviposition period conform to homogeneity of variance and normal distribution, the data of the other reproductive parameters of the females did not simultaneously meet these 2 conditions, so non-parametric single factor analysis Kruskal-Wallis rank test was used to compare the influence of pressure given in different periods on the ovipositing periods of the prey. All the pairwise comparisons were an-alyzed using the Wilcoxon rank test. Because there are 2 levels (sex and predation-pressure) of the variance, Cox proportional-hazards model were used to analyze the survival curve, followed by Kaplan-Meier survival analysis with log-rank test to compare the survival curve in pairs. Because the data of lifespan were also not normally distributed, correlations between lifespan and fecundity, and lifespan and body size were analyzed by using Spearman's rank correlation method.

Immature survival and development
Immature survival rates were high for all groups (each started with 50 replicates): 47 (24 females and 23 males) prey mites developed to the adults successfully in the control group; 47 (26 females and 21 males) in the L group; 46 (26 females and 20 males) in the P group; 45 (23 females and 22 males) in the T group; 47 (26 females and 21 males) in the Ovi group, and 45 (22 females and 23 males) in the Full group. The survival rates were not significant different among treatments (χ 2 = 1.570, df = 5, P = 0.905). As only 1 immature individual did not successfully develop to adult in the P group, hatching rates were the same as the survival rates statistically.
The developmental time of the T. putrescentiae was significantly affected by predation stress (df = 5, H = 118.474, P < 0.001) and was different for the 2 sexes (df = 1, H = 36.933, P < 0.001; Table 1), while the interaction between treatment and sex was not significant (df = 5, H = 5.563, P = 0.351). Prey mites of the Full group (exposure to predation stress whole lifespan) showed the longest developmental time among all the groups (all P < 0.001; Fig. 1A), whereas the individuals of the 3 groups (Control, T, and Ovi) had similar developmental times (all P > 0.05), which were shorter than the other 2 groups (L and P groups) (except Ovi and P : W = 830.5, P = 0.035, all P < 0.001). The L group (delayed by 12.7%) and the P group (delayed by 4.8%) took similar a length of time to develop to adults (W = 1187.5, P = 0.288). Overall, T. putrescentiae females had longer developmental time than the males (W = 13205, P < 0.001).
Further comparisons of each stage (Fig. 1B) showed that although the mites in T group and the Full group had longer tritonymphal periods, there were no statistical differences in the durations of the egg period and tritonymphal period (all P > 0.05). The control, P, T, and the Ovi groups had similar developmental times in the larval period (all P > 0.05) and their larvae periods were all shorter than the larvae period of the L group (all P < 0.001). In addition, the control, L, T, and the Ovi groups had similar developmental time in the protonymphal period (all P > 0.05), and their protonymphal periods were all shorter than the protonymphal period of the P group (all P < 0.001). Moreover, the individuals of the Full group took the longest time in the larval period and protonymphal period (all P < 0.001).

Survival and lifespan
For males, the results were consistent with the results of comparisons among all individuals ( Fig. 2A). To be specific, the L and the P groups had similar highest survival rates (χ 2 = 0.2, df = 1, P = 0.6) (pairwise comparisons with other groups: all P < 0.05). The Control group had a similar survival rate with the Ovi group (χ 2 = 0.1, df = 1, P = 0.8), while their survival rates were higher than the T group (all P < 0.05). Moreover, the males of the full group had the lowest survival rates (all P < 0.001).
For females, the L, P, and Ovi groups had similar survival rates (all P ≥ 0.2), while the control and the T group had similar survival rates (χ 2 = 1.8, df = 1, P = 0.2) (Fig. 2B). Furthermore, the former 3 groups (L, P, Ovi) had higher survival rates than the latter 2 groups (control and T) (all P < 0.005), while the females of the full group had the lowest survival rates (all P < 0.001).
Comparisons of survival curves among treatments for all individuals (Fig. 2C) revealed that those of the L and the P groups were similar (χ 2 = 0.3, df = 1, P = 0.6), while both showed higher survival rates than the control group (L: χ 2 = 17.5, df = 1, P = 0.000; P: χ 2 = 16.7, df = 1, P < 0.001 but were similar to survival rates with the Ovi group (χ 2 = 1.6, df = 1, P = 0.2). In addition, the individuals of the T group had slightly lower survival rates than the individuals of the control (χ 2 = 4.4, df = 1, P = 0.04). The Full group had the lowest survival rates (all P < 0.001).
The lifespan analysis indicated that treatment and sex had significant effects on the prey (all P < 0.001); however, the 2 factors did not have interactions (df = 5, H = 7.193, P = 0.207). Males lived longer than the females (W = 4080, P < 0.001, Fig. 3), similar to trends in the results of the survival rates. Specifically, the individuals of the L and the P groups lived the longest (W = 815.5, P = 0.731) (pairwise comparisons with other groups: all P < 0.05). The individuals of the Ovi group and Control group had similar lengths of lifespan (W = 818.5, P = 0.219), and the individuals of these 2 groups lived longer than the individuals of the T group (all P < 0.05). Individuals of the Full group had the shortest lifespan (all P < 0.001).

Female reproductive parameters
Except for the pre-oviposition period (χ 2 = 2.343, df = 5, P = 0.799), all other female reproductive parameters were significantly affected by treatment (all P < 0.001; Table 2). To be specific, the oviposition periods of the control, L, and T groups were similar (all P > 0.1), while the oviposition periods of the L, P, and Ovi groups were similar (all P > 0.1). However, the females of the T group had shorter oviposition periods than the females of the P group (W = 87.50, P = 0.005) and the Ovi group (W = 86, P = 0.003). In addition, the females of the control group had slightly shorter oviposition periods than the females of the P group (W = 114.5, P = 0.025) and the Ovi group (W = 118, P = 0.02), whereas the Full group had the shortest oviposition period (all P < 0.001). The control, L, P, and the Ovi groups had similar durations of the post-oviposition period (all P > 0.1), while the post-oviposition periods of the control group and the T group had similar length (W = 188, P = 0.711). However, the post-oviposition period of the T group was only slightly shorter than the post-oviposition periods of the L, P, and Ovi groups (all P < 0.05). The females of the full group lived the shortest time after laying eggs (all P < 0.001). Table 3 The females of the control group and the T group had similar reproductive rates (W = 293, P = 0.618), while the L group and the P group had similar reproductive rates as well (W = 249.5, P = 0.618). Furthermore, the former 2 groups (the control and the T groups) had higher reproductive rates than the latter 2 groups (the L and the P group), while these 4 groups all had greater reproductive rates than the Ovi group (all P < 0.05). The females of the Full group laid the least number of eggs per day (all P < 0.000).
The females of the T group laid similar number of total eggs to the females of the control group (W = 208, P = 0.312), while the L group and the Ovi group had similar number of total eggs (W = 138.5, P = 0.08). Furthermore, the females of the control and the T groups laid more eggs in total than the females of the P group, while the females of the P group laid more eggs in total than the

<0.001
Note: Means (± se) in the same row with same letters are not statistically different at P = 0.05.  females of the L and the Ovi groups (all P < 0.001). The females of the full group had the lowest fecundity during the oviposition period (all P < 0.001).

Correlations between lifespan, fecundity, and body size
The correlation between fecundity and lifespan was not significant (P = 0.067; Fig. 4); however, body size was inversely correlated with lifespan when male and female data were pooled (P < 0.001, rho = −0.339; Fig. 5), suggesting bigger females having shorter lifespan than the males in each group (P < 0.001). However, the body size of males did not correlate with the lifespan (P = 0.531); similar results were found in females (P = 0.554). Moreover, treatment did not show influences on body size (P = 0.996; Table 3).

Discussion
To test the hypothesis of  that milder levels of predation stress might have no effect on prey lifespan or even increase it,  reduced the duration of exposure to predation stress and showed that predation stress applied only during immature development could increase lifespan (by 9.7%), although, it is not clear if the benefits of this "milder" predation stress resulted from the short duration of exposure or from the early life exposure. This study therefore further reduced the duration of exposure to predation pressure during the early part of prey life history and demonstrated that only the very early stages (larva and protonymph) responded positively to predation stress and therefore extended lifespan.

Lifespan and survival rates
In this study, we applied predation stress to each active immature stages (larva, protonymph and tritonymph) and the first 5 d of oviposition period of the adult prey. This short duration of exposure accounts for merely a small proportion (more or less 5%) of the lifespan of the mite (for comparison, we also prepared a treatment where mites were exposed to predation stress during full lifespan). We showed that predation stress during larval or protonymphal stage actually increased lifespan by 6.6% and 6.4% in males, and 9.5% and 8.7% in females, respectively, whereas exposure during tritonymphal stage reduced lifespan slightly by 4.5% in males and 2.2% in females. Exposure during the first 5 d of the oviposition period had no effect on prey lifespan, despite the fact that larval, protonymphal or tritonymphal stage is shorter than 5 d. In comparison, exposure of predation stress for the full life reduced lifespan dramatically by 30.3% in males and 28.9% in females. Our previous study  also examined the effects of predation stress over the whole lifespan of the prey; and predation stress by 5 predators resulted in a reduction of lifespan by 34.3% in males and 30.1% in females, which agrees with the results of this study. Another of our studies (Wei et al., 2022 in preparation) showed that predation stress given during the entire immature period (about 12-16 d) increased the lifespan of both male and female by 9.7% on average, whereas prey exposure to predation stress  during their oviposition and post-oviposition periods reduced their lifespans by 24.8% and 28.7%, respectively. The patterns in survival rates among different treatments in our studies showed broadly similar trends in data on lifespan. Although the prey in L and P groups had increased lifespans and survival rates, the lifespan and sur-vival rate of the prey in the T group decreased slightly. This cannot be explained by shorter exposure ("milder" stress) being able to generate opposite effects on the prey. Taken together, these showed (1) stage-dependency: there were contrasting effects of predation stress on lifespan in different life stages of prey (i.e.); the very early life stages (larva and protonymph) responded positively to predation stress with extended lifespan, whereas tritonymphal and adult stages responded negatively with reduced lifespan, with the degree of responses stronger in post-oviposition period; (2) duration dependency: the duration of exposure can be important for adults, for example, for the oviposition period when short exposure to predation stress for 5 d had no effect on lifespan.
Although there have been only a few studies on the effects of predation stress on prey lifespan and aging, similar lines of research examining other factors such heat/cold stress are well known. For example, extreme nonlethal chilling and heat could decrease the lifespans of females (Søvik & Leinaas, 2003;Jiao et al., 2016;Zhang et al., 2016), but repeated transient heat or fluctuated heat and cold effects were relatively mild, and could have positive effects on lifespans (Hercus et al., 2003;Søvik & Leinaas, 2003;Torson et al., 2015;Zheng et al., 2017). Also, repeat short-period fasting (relatively mild) was known to be positive to lifespans (Varady & Hellerstein, 2007;Colman et al., 2009Colman et al., , 2014Singh et al., 2012;Caramoci et al., 2016;Li & Zhang, 2019b). However, different factors can influence lifespan in different mechanisms. For example, temperature usually influences organisms by manipulating the expression of the heat shock proteins (e.g., Hercus et al., 2003;Sørensen & Kristensen, 2007;Zhu et al., 2017), while diet restriction may influence the metabolic rate and related-signaling, and reduce free radical production (Fontana et al., 2010;Mcdonald & Ramsey, 2010). Predation stress is very complex as it can influence many aspects of organisms and induce changes through many different internal mechanisms, like neurochemistry (Nanda et al. 2008), gene expression (Zhang et al., 2009), amygdala (Choi & Kim, 2010), and glucocorticoid level (Creel et al., 2009;Thaler et al., 2012).

Immature development
In this study, the prey in the L and P groups delayed their developments by 12.73% and 4.83%, respectively; those in the T group showed no change in development time; but those in the Full group had a dramatic increase in developmental time (18.25%). Further analysis of each life stage revealed that predation stress received during the larval stage only extended the duration of the larva, and similar predation stress received during the protonymphal stage only extended the duration of the protonymph (Fig. 1B); although the duration of subsequent immature stages was not increased, adult longevity was increased. This is quite different from the compensatory effects of transient intraguild predation (IGP) risk on the early life of Phytoseiulus persimilis: the mites exposed to IGP risk during larval stage delayed larval development but accelerated the development the protonymphal stage (Walzer et al., 2015).
This study showed that increase in development time could result in increased lifespan (in L & P groups) or reduced lifespan (Full groups), not in agreement with the tradeoff "live fast, die young" (Hunt et al., 2004;Monaghan et al., 2009;Bestion et al., 2015;Travers et al., 2015;Hooper et al., 2017). This may suggest that the trade-off between early and late life traits is complex, and this prey may fail to balance the allocation of the resource under high predation stress.
Previous studies had showed that under stress prey usually developed fast and matured at a relatively smaller size (McPeek et al., 2001;Altwegg, 2002;Peckarsky et al., 2002;Dahl & Peckarsky, 2003;Griffis-Kyle & Ritchie, 2007;Thaler et al., 2012;Clinchy et al., 2013). In this study, the prey did not show any differences in body size among different treatments, a result consistent with results found in spider mites (Li & Zhang, 2019a).

Reproduction
Our study showed that predation stress reduced fecundity by 19.15%, 15.43%, 21.27%, and 48.65% in the L, the P, the Ovi, and the Full groups, respectively. Among them, the 3 former groups reduced fecundity by reducing daily reproduction, while the last group did so by decreasing both daily reproduction and the oviposition period. The T group had no difference in reproductive parameters with the control. The L and P groups had longer lifespans and lower reproductive rates, which was consistent with previous studies that suggested there were trade-offs between reproduction and lifespan (Adler et al., 2013(Adler et al., , 2016Rodríguez-Muñoz et al., 2019). However, the Full group which had a significant drop in fecundity and also had a dramatic reduction of lifespan, was the opposite of the L and P groups, but was consistent with the study on spider mites that showed that female mites could have a reduction in both fecundity and lifespan (Li & Zhang, 2019a). Moreover, the Ovi group showed decreased fecundity, but similar lifespan, while the T group showed similar fecundity, but slightly shorter lifespan. This may indicate that there could have been a trade-off between early life development and reproduction and late life lifespan under relatively mild stress, because while both the L and P groups had delayed development and fecundity, they had prolonged lifespan. While the Ovi group only had a reduction in fecundity, it was not sufficient to extend lifespan-thus it had similar lifespan with the control. The T group did not have any difference in either development or fecundity, and this had resulted in a slightly decrease in the lifespan. However, the high predation stress given in the Full group prevented mites to balance the allocation-thus this treatment did not show such trade-offs.

Sex differences
Prey response to predation stress in this study also showed sex-specific differences. Males developed faster, matured in smaller sizes and lived longer than females. This study confirmed previous studies that males could develop faster to defend predators and secure more mating opportunities, while females could mature larger to ensure reproductive quality (De Block & Stoks, 2003;Mikolajewski et al., 2005). Although among different treatments, lifespan trends were similar in both males and females, the survival rates of females are slightly different from those of males. To be specific, the females in the Ovi group had higher survival rates than the control group, while the males had similar survival rates. Our results confirmed that females seem to be more sensitive to predation stress (Walzer & Schausberger, 2011), especially during the oviposition period. The response of females to the mild stress supplied during the oviposition period generated benefits to their survival rates.

Conclusion
The most significant conclusion from this study is that the effects of predation stress are prey-stage-dependent and early life experience is most important to lifespan modulation. The first 2 stages (the larva and protonymph), although very short (each about 5% of the lifespan), were most sensitive to predation stress, which prolonged immature development, decreased fecundity but extended lifespan; the tritonymph stage was the least sensitive to predation stress, which had no effect on immature development and fecundity but slightly reduced lifespan; the first 5 d of the oviposition period was sensitive to predation stress, which decreased fecundity but not lifespan. It will be worthwhile exploring the effects on lifespan of even shorter-term exposure to predation stress (e.g., part of larval or protonymphal period or even a single day) in future studies.
for laboratory assistance. Anne Austin (Manaaki Whenua -Landcare Research, Palmerston North, New Zealand) kindly review the draft of this manuscript and provided useful suggestions. Zhi-Qiang Zhang was supported in part by New Zealand Government core funding for Crown Research Institutes from the Ministry of Business, Innovation and Employment's Science and Innovation Group.
Open access publishing facilitated by The University of Auckland, as part of the Wiley -The University of Auckland agreement via the Council of Australian University Librarians.

Disclosure
The authors declare that there is no conflict of interests.