An inverse Lansing effect: Older mothers produce offspring with improved development and growth efficiency

13 The Lansing effect predicts a decline in offspring performance with increasing maternal age. 14 Maternal age and diet can influence offspring development and fitness via maternal effects, 15 but how these two factors interact remains poorly understood. We examined how maternal 16 age at oviposition and dietary conditions affect offspring developmental plasticity in a 17 thelytokous predatory mite (Amblyseius herbicolus). Mothers were provided either a 18 restricted or abundant prey diet, and their offspring were exposed to varying prey availability 19 and monitored for hatching success, survival to adulthood, developmental time, size at 20 maturity, and prey consumption. We addressed two main questions: How does maternal age 21 affect offspring developmental time and size at maturity and does maternal diet modify the 22 effect of maternal age on offspring? Our results suggest an inverse Lansing effect. Offspring 23 of older mothers showed increased survival and reduced prey consumption without any 24 compromise in terms of size at maturity. Interactions were found between maternal diet and 25 age on offspring prey consumption and developmental plasticity. Notably, offspring from 26 older, diet-restricted mothers achieved the best overall performance during development. Our 27 study demonstrates that maternal age and diet jointly shape offspring development, and 28 highlights the importance of incorporating maternal age into studies of maternal effects and 29 phenotypic plasticity. 30

Acari, developmental plasticity, maternal effect, phenotypic plasticity, 31 Phytoseiidae, transgenerational phenotypic plasticity 32 33 1 Introduction 34 To cope with environmental variability, organisms have evolved diverse strategies to 35 optimise survival and reproduction (West-Eberhard, 1989). One such strategy is phenotypic 36 plasticity, which is defined as the ability of a single genotype to produce different phenotypes 37 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 3 in response to environmental variation (West-Eberhard, 2003). Phenotypic adjustments can 38 occur during an organism’s development, a process known as developmental plasticity, 39 which often results in irreversible trait changes (Piersma & Drent, 2003; West-Eberhard, 40 2005; Blanckenhorn, 2009; Taborsky, 2017). Developmental plasticity allows individuals to 41 adjust life-history traits such as growth rate, development time, and size at maturity, 42 particularly under suboptimal conditions such as food limitation or temperature stress 43 (Stearns & Koella, 1986; Blanckenhorn, 1999; Bize et al., 2003). This ability is especially 44 important in species with determinate growth, where development ceases upon maturity (Bize 45 et al., 2003). 46 However, plastic responses are not confined to an individual’s own generation 47 (Räsänen & Kruuk, 2007; Bonduriansky & Head, 2007). Increasing evidence shows that 48 parental environmental experiences can shape offspring phenotypes via maternal and/or 49 paternal effects, a form of non-genetic inheritance that is widespread across taxa 50 (Bonduriansky & Day, 2009; Bonduriansky, 2012; Luquet & Tariel, 2016; Kuijper & 51 Johnstone, 2018; Bell & Hellmann, 2019). Through mechanisms such as epigenetic 52 modification, hormonal signalling, or differential provisioning, parents can influence 53 offspring traits including body size, development, stress tolerance, and survival 54 (Bonduriansky & Day, 2009; Bonduriansky, 2012; Luquet & Tariel, 2016; Kuijper & 55 Johnstone, 2018; Bell & Hellmann, 2019; Groothuis et al., 2020). Parental effects can also 56 alter offspring developmental trajectories (Plaistow et al., 2004; Luquet & Tariel, 2016; 57 Groothuis et al., 2020). 58 Among intrinsic parental factors, maternal age has emerged as a key determinant of 59 offspring phenotype (Plaistow et al., 2015; Perez et al., 2017; Bonduriansky et al., 2018; 60 Hernández et al., 2020). The decline in offspring performance with maternal age—known as 61 the Lansing effect or maternal effect senescence—has been found across a wide range of 62 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 4 taxa, from rotifers and insects to birds and mammals (Lansing, 1947; Fox et al., 2003; 63 Bouwhuis et al., 2015; Schroeder et al., 2015; Moorad & Nussey, 2016; Reichert et al., 2020; 64 Krug et al., 2020; Ivimey-Cook et al., 2023). Maternal age effects can manifest as reduced 65 hatching success, lower stress resistance, smaller size, or shortened lifespan in offspring 66 (Halle et al., 2015; Bloch Qazi et al., 2017; Hernández et al., 2020). The adverse influences 67 of advanced maternal age are often context-dependent and shaped by environmental 68 conditions such as diet (Plaistow & Benton, 2009; Gribble et al., 2014; van Daalen et al., 69 2022). 70 Two main theoretical frameworks have been put forward to explain maternal age 71 effects. The senescent parent hypothesis proposes that physiological deterioration reduces 72 gamete quality or maternal provisioning, thereby reducing offspring fitness (Kong et al., 73 2012; Plaistow et al., 2015; Bloch Qazi et al., 2017). For example, maternal age is linked to 74 oxidative damage in Drosophila oocytes (Fredriksson et al., 2012) and reduced nutrient 75 provisioning in eggs of the wasp Eupelmus vuilletti (Muller et al. 2017). Such findings 76 challenge the traditional separation between germline and soma, suggesting that age-related 77 decline can penetrate the Weismann barrier (Monaghan & Metcalfe, 2019). By contrast, 78 adaptive hypotheses predict age-related shifts in reproductive strategy (Goos et al. 2019). 79 Both the asset protection principle and the reproductive restraint hypothesis suggest that older 80 individuals may reduce reproductive effort and increase investment in somatic maintenance 81 (Clark, 1994; Jehan et al., 2021). Conversely, the terminal investment hypothesis suggests 82 increased reproductive effort late in life (Monaghan et al., 2020; Jehan et al., 2021). For 83 instance, young soil mite (Sancassania berlesei) mothers produce more but smaller eggs, 84 whereas older mothers produce fewer but larger ones (Plaistow et al., 2007). 85 Crucially, the strength and direction of maternal age effects often depend on 86 environmental conditions, especially diet (Rollinson & Hutchings, 2013; van Daalen et al., 87 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 5 2022). Diet stress can exacerbate negative effects on offspring (Vijendravarma et al., 2010; 88 Hafer et al., 2011), whereas mild diet restriction (i.e., caloric restriction) may extend maternal 89 lifespan and sometimes even enhance offspring performance (Gribble et al., 2014; Hibshman 90 et al., 2016). Interactions between maternal age and diet are predicted to strongly influence 91 offspring developmental plasticity (van den Heuvel et al., 2016). 92 Despite growing recognition of these interactions, empirical studies remain scarce. To 93 address this, we investigated the combined effects of maternal age and diet on the offspring 94 of a thelytokous predatory mite, Amblyseius herbicolus (Chant) (Acari: Phytoseiidae). This 95 asexual, oviparous species provides a system that eliminates confounding factors such as 96 paternal genetic input and maternal care, enabling a clear focus on physiological and 97 epigenetic maternal effects (Castonguay & Angers, 2012; Verhoeven & Preite, 2014; 98 Groothuis et al., 2020; Vogt, 2021). Although no Lansing effect was previously detected in 99 A. herbicolus when fed ad libitum prey, older females consistently produced smaller eggs, 100 suggesting age-related shifts in provisioning (Zhang et al., 2024). How these shifts influence 101 offspring developmental plasticity remains unclear. 102 Here, we tested two hypotheses: 103 1. Offspring of older mothers exhibit reduced developmental plasticity, expressed as 104 narrower ranges of developmental time and body size at maturity. 105 2. Dietary restriction modulates maternal age effects, strengthening negative age effects 106 under low-resource conditions. 107 By examining the interaction between maternal condition and offspring 108 developmental plasticity, this study provides insights into our understanding of 109 transgenerational effects and their ecological and evolutionary significance, especially in 110 asexually reproducing species. 111 112 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 6 2 Materials and Methods 113 2.1 Study animals and feeding conditions 114 Amblyseius herbicolus is a generalist predatory mite, typically less than 500 µm in body 115 length (McMurtry et al., 2013; Zhang & Zhang, 2021). It has five life stages (egg, larva, 116 protonymph, deutonymph, and adult; FIGURE S1), a developmental period of about 1 week, 117 and a lifespan of about 1 month under laboratory conditions (Liu et al., 2024a). We used a 118 laboratory population of A. herbicolus to examine the influence of maternal age and diet on 119 offspring developmental plasticity. The founding population (>30 adult females) was 120 collected from avocado (Persea americana Mill.) leaves in an orchard in Te Puna, Tauranga, 121 New Zealand. Species identification was confirmed based on morphological characteristics 122 described by Ma et al. (2024). After collection, predatory mites were fed the dried fruit mite 123 Carpoglyphus lactis L. (Acari: Carpoglyphidae), sourced from Bioforce Limited (Karaka, 124 Auckland, New Zealand), for 3 months before the experiment (approximately six 125 generations). Colonies were maintained on water-filled plates (Figure S2A; Zhang & Zhang, 126 2021) within plexiglass cabinets at 25 °C ± 1 °C, 80% ± 5% relative humidity, and a 16:8 h 127 (light:dark) photoperiod. 128 129 2.2 Preparing prey eggs 130 Frozen C. lactis eggs were used as prey. These eggs are suitable food for predatory mites 131 (Liu et al., 2024a). Using non-viable eggs reduced contamination from yeast-based rearing 132 and the need for frequent cell changes. Eggs of C. lactis were collected following Liu et al. 133 (2024b), frozen at -18 °C for about a week, thawed at room temperature for 20 min, and used 134 within 1 month of freezing. 135 136 2.3 Experimental procedures 137 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 7 2.3.1 Maternal generation 138 Approximately 50 A. herbicolus eggs (<16 h old) were transferred to new plates 139 (FIGURE S2A) and reared with ad libitum access to mixed-stage C. lactis. Eggs were 140 collected by placing 1 cm sewing threads in colonies overnight, producing synchronous 141 cohorts. Development to adulthood requires ~10 days, so new threads were placed on days 142 10–15 to collect newly laid eggs (<16 h old). Threads were replaced daily, and collected eggs 143 were used in experiments (FIGURE 1A). 144 145 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 8 146 FIGURE 1. Schematic representation of the experimental design used in this study. A: 147 Experimental setup for examining the effects of maternal age and dietary conditions on the 148 developmental plasticity of Amblyseius herbicolus offspring. Sample sizes (N) are provided 149 for each treatment group; for daughters, sample sizes are shown in brackets, with values for 150 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 9 restricted and abundant maternal diets listed on the left and right, respectively. B: Feeding 151 schedule (feeds 1–5) during the immature development of A. herbicolus for both mothers and 152 daughters. Since the hatching time of A. herbicolus eggs exceeded 24 h, the first feeding 153 occurred on day 2 to ensure newly hatched larvae could access food. To prevent an 154 accumulation of uneaten prey before larval emergence, the second feeding was administered 155 only after larvae were observed, typically on day 3. Subsequent feedings continued daily until 156 the final prey provision. 157 158 These synchronous cohorts of A. herbicolus eggs were randomly assigned to one of 159 two dietary treatments and reared individually until oviposition (FIGURE S2B), as follows: 160 1. Restricted diet: 30 prey eggs in total during immature development (6 per day for 5 days; 161 FIGURE 1B), and 15 eggs per day during adulthood. 162 2. Abundant diet: 50 prey eggs in total during immature development (10 per day for 5 days; 163 FIGURE 1B), and 40 eggs per day during adulthood. 164 Twenty eggs were allocated to each treatment group as replicates. Reproduction is 165 thelytokous so mating was not required. Upon maturity, females were transferred to new 166 rearing cells, and prey was replenished daily to facilitate oviposition. 167 168 2.3.2 Offspring generation 169 Eggs (<16 h old) laid by mothers were assigned to two measurements: 170 1. Egg size measurement: egg volume (V) was estimated from length (L) and maximum 171 breadth (B) using Narushin’s (2005) formula: V = (0.6057 - 0.0018B)LB2 172 2. Developmental plasticity: daughters were reared individually (FIGURE 1A) and provided 173 with one of five prey densities during immature development: 4, 6, 8, 10, or 12 eggs per 174 day for 5 days (20–60 eggs total; FIGURE 1B). Offspring were classified by maternal age 175 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 10 at oviposition as ‘young’ or ‘old,’ with the threshold defined as half the mean oviposition 176 period of mothers in each dietary treatment group. 177 178 2.4 Data collection 179 Both the mothers and daughters were monitored twice daily (08:00 and 17:30). For mothers, 180 developmental time (egg to adult), prey consumption (number of prey eggs consumed), size 181 at maturity (dorsal plate length), fecundity (lifetime oviposition), and lifespan (egg to death) 182 were recorded. For offspring, hatching success, survival to adulthood, developmental time, 183 prey consumption, and size at maturity were recorded. Adult size for both generations was 184 determined by mounting mature individuals in Hoyer’s medium and examining them under a 185 compound microscope (Walter & Krantz 2009). 186 187 2.5 Statistical analysis 188 Analyses were conducted in R (R Core Team, 2024) in RStudio (version 2024.09.1). 189 Graphics and models were generated using ggplot2 and lme4 (Bates et al., 2015; Wickham, 190 2016). For the maternal generation, two eggs from the abundant group did not hatch, and one 191 individual from the abundant group and two from the restricted group were lost during 192 feeding. The remaining individuals survived to oviposition and were analysed. Maternal life-193 history traits were summarised as means ± standard errors of the mean (SEMs). Generalised 194 linear models (GLMs) with Poisson or quasi-Poisson distributions tested the effect of dietary 195 treatments, with model dispersion verified post hoc. Spearman’s correlations examined the 196 relationships between maternal age, prey consumption, and daily oviposition. Egg volume 197 was analysed with linear mixed-effects models (LMMs), including maternal diet and age as 198 fixed effects and mother as a random effect. 199 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 11 For the offspring generation, logistic regressions tested the effects of maternal diet, 200 maternal age, and offspring prey availability on hatching and survival (binomial response). 201 Developmental time and size at maturity were analysed using LMMs with fixed factors 202 (maternal diet, maternal age, and prey availability), a covariate (prey consumption), and 203 mother as a random factor. Likelihood ratio tests (LRTs) were used to assess model fit. 204 Preliminary models including prey availability showed no significant effect on developmental 205 time (LRT: c² = 4.258, df = 4, P = 0.372) and size at maturity (χ² = 6.174, df = 4, P = 0.187), 206 and prey availability was therefore excluded from the final models. Pearson’s correlations 207 assessed the relationships between prey consumption and developmental traits. Offspring 208 prey consumption was analysed using a Poisson GLM, with maternal diet and age as fixed 209 factors and prey consumption as a covariate. Statistical significance was set at α = 0.05. 210 211 3 RESULTS 212 3.1 Diet-induced influences on maternal parameters 213 Several life-history traits of mothers were significantly influenced by dietary conditions 214 (TABLE S1). Individuals in the restricted diet group exhibited longer developmental and 215 oviposition periods and an extended lifespan, whereas those in the abundant diet group 216 showed higher daily oviposition rates, greater maximum daily fecundity, and larger body 217 size. The post-oviposition period and lifetime fecundity did not differ between treatments, 218 while the pre-oviposition period was marginally non-significant (TABLE S1). 219 Individuals given more prey consumed more during both immature development and 220 adulthood (TABLE S1). After reaching adulthood, daily prey consumption declined with age 221 in both dietary groups (Figure S3). Average daily oviposition ranged from one to two eggs in 222 both treatment groups; in the restricted group, this rate remained consistent throughout the 223 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 12 oviposition period, whereas in the abundant group it declined with maternal age 224 (FIGURE S4). 225 Egg size was significantly influenced by dietary condition (LMM: Wald c² = 57.673, 226 df = 1, P < 0.001) and maternal age at oviposition (Wald c² = 74.691, df = 1, P < 0.001). 227 Mothers on the abundant diet produced larger eggs, and egg size declined with age. A 228 significant interaction between diet and maternal age (Wald c² = 47.163, df = 1, P < 0.001) 229 indicated that egg size decreased more sharply with age in mothers on the restricted diet 230 (FIGURE 2). 231 232 233 FIGURE 2. Egg size (volume) of Amblyseius herbicolus as influenced by maternal diet 234 (restricted or abundant) and age at oviposition. Solid lines indicate regression fits, with 235 shaded areas showing 95% confidence intervals. Regression equation, Pearson’s correlation 236 coefficient (R), and P-value are provided. 237 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 13 238 3.2 Maternal diet and age influences on offspring parameters 239 3.2.1 Hatching and survival to adulthood 240 Offspring were classified by maternal age at oviposition based on mean oviposition periods 241 (TABLE S1). In the restricted group, ‘young’ offspring were derived from eggs laid on days 242 1 to 18, and ‘old’ from day 19 onwards; in the abundant group, ‘young’ were from days 1 to 243 13, and ‘old’ from day 14 onwards. 244 Hatching success was unaffected by maternal diet (logistic regression: Wald 245 c² = 0.001, df = 1, P = 0.972), maternal age at oviposition (Wald c² = 0.576, df = 1, 246 P = 0.448), offspring diet (Wald c² = 0.269, df = 1, P = 0.604), or their interactions 247 (FIGURE 3A). In contrast, survival to adulthood was influenced by offspring diet (Wald 248 c² = 83.683, df = 1, P < 0.001) and maternal age (Wald c² = 7.040, df = 1, P = 0.008), with a 249 significant interaction between maternal age and offspring diet (Wald c² = 9.463, df = 1, 250 P = 0.002). Specifically, offspring from older mothers and those provided more prey had 251 higher survival, particularly under low prey availability (FIGURE 3B). 252 253 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 14 254 FIGURE 3. Hatching rate (A) and survival to adulthood (B) of Amblyseius herbicolus 255 offspring under different offspring prey diets (20–60 eggs) and maternal age/diet treatments. 256 Panels separate restricted and abundant maternal diets. Offspring from young and old 257 mothers are defined as in the text. 258 259 3.2.2 Developmental duration and size at maturity 260 Developmental duration was significantly affected by maternal age (LMM: 261 Wald c² = 38.063, df = 1, P < 0.001) with a significant interaction between maternal age and 262 diet (Wald c² = 21.710, df = 1, P < 0.001), but not maternal diet (Wald c2 = 0.878, df = 1, 263 P = 0.349) and prey consumption (Wald c2 = 1.122, df = 1, P = 0.290). Offspring from older 264 mothers took longer to mature, particularly in the abundant diet group (FIGURE 4). 265 266 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 15 267 FIGURE 4. Developmental duration of offspring in relation to prey consumption, maternal 268 diet, and maternal age at oviposition. Solid lines show regression fits; shaded areas indicate 269 95% confidence intervals. Offspring from young and old mothers are defined as in the text. 270 271 Size at maturity was significantly influenced by prey consumption (LMM: Wald 272 c² = 56.224, df = 1, P < 0.001), maternal age (Wald c² = 4.238, df = 1, P = 0.040), and the 273 interaction between maternal diet and age (Wald c² = 4.167, df = 1, P = 0.041). Larger size 274 was associated with higher prey consumption and older mothers; differences between 275 offspring of young and old mothers were more pronounced in the restricted maternal diet 276 group (FIGURE 5). 277 278 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 16 279 FIGURE 5. Dorsal plate length of offspring in relation to prey consumption, maternal diet, 280 and maternal diet. Solid lines show regression fits; shaded areas indicate 95% confidence 281 intervals. Offspring from young and old mothers are defined as in the text. 282 283 3.2.3 Prey consumption 284 Offspring prey consumption increased with prey availability (GLM: Wald c2 = 859.229, 285 df = 1, P < 0.001). Maternal age at oviposition (Wald c2 = 30.510, df = 1, P < 0.001) and the 286 interaction between maternal diet and age (Wald c2 = 4.288, df = 1, P = 0.038) were also 287 significant: offspring of older mothers consumed less, especially when mothers were on the 288 restricted diet (FIGURE 6). Maternal diet alone had no significant effect (Wald c2 = 1.401, 289 df = 1, P = 0.236). 290 291 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 17 292 FIGURE 6. Offspring prey consumption by prey availability (20, 30, 40, 50, and 60 eggs), 293 maternal diet, and maternal age. Individual points show observations (beeswarm); boxplots 294 indicate median, interquartile range (IQR), and 1.5 × IQR whiskers. Panels separate restricted 295 and abundant maternal diets. Sample sizes (n) are indicated below each box. Offspring from 296 young and old mothers are defined as in the text. 297 298 4 Discussion 299 Our study provides evidence for an inverse Lansing effect in the predatory mite 300 A. herbicolus. Offspring of older mothers showed higher survival to maturity, longer 301 development, larger body size, and reduced prey consumption during development. 302 Moreover, offspring of older mothers exhibited no significant reduction in developmental 303 plasticity compared to those of younger mothers. These findings contrast with the classical 304 Lansing effect and our first hypothesis, but align with studies showing that late-life 305 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 18 reproduction can result in enhanced offspring quality in some species (Marshall et al., 2010; 306 Travers et al., 2021; Anderson et al., 2022). For instance, older spiders (Argiope radon) 307 produce offspring with greater starvation tolerance (Ameri et al., 2019), and beetles 308 (Menochilus sexmaculatus) develop faster when produced by older mothers (Singh et al., 309 2021). Such findings suggest that maternal age effects may represent adaptive shifts in 310 reproductive strategy rather than simple deterioration. Our results indicate a terminal 311 investment strategy rather than senescent decline in A. herbicolus. 312 313 4.1 Maternal investment and offspring performance 314 Maternal diet strongly influenced life-history traits in A. herbicolus. Females on a restricted 315 diet produced fewer eggs per day but extended their oviposition period and lived longer, 316 resulting in lifetime fecundity comparable to females on an abundant diet. This trade-off 317 between current reproduction and survival is consistent with resource-allocation models 318 observed across taxa (Kaitala, 1991; Zera & Harshman, 2001; Lee et al., 2020). 319 Egg size and number were affected by both maternal diet and age. Larger eggs were 320 produced under the abundant diet, which is consistent with other predatory mite species 321 (Walzer & Schausberger, 2015). However, the reduction in egg size and number with 322 advanced maternal age was only observed in those given the abundant diet. Such age-related 323 patterns vary across taxa, with decreases in egg size in Drosophila melanogaster (Bloch Qazi 324 et al., 2017) but increases in ladybirds (Coleomegilla maculata) (Vargas et al., 2012). 325 Interestingly, diet-restricted females showed more stable investment across their reproductive 326 lifespan, and we found no evidence for a size–number trade-off as observed in other species 327 (Gliwicz & Guisande, 1992). 328 Despite reductions in egg size with maternal age, offspring performance was not 329 compromised. Larger eggs are typically associated with enhanced offspring growth, size, and 330 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 19 survival (Fox, 1994; Dias & Marshall, 2010; Rius et al., 2010). Thus, our finding suggests 331 that maternal age effects in A. herbicolus operate independently of egg size. Similar findings 332 have been observed in the soil mite S. berlesei (Benton et al., 2008). Since egg provisioning 333 is an important mechanism underlying maternal age effects (Mousseau & Dingle, 1991; 334 Mousseau & Fox, 1998; Yanchula & Alto, 2021), egg nutrient content and composition, 335 rather than size alone, may be key factors in A. herbicolus. For example, older mothers of the 336 parasitic wasp Eupelmus vuilleti showed reduced egg provisioning with protein, sugar, and 337 lipids, which resulted in a lower nutrient composition at maturity (Muller et al., 2017). 338 Testing whether maternal age alters egg nutrient composition in A. herbicolus remains an 339 important future direction for research. 340 341 4.2 Maternal diet-by-age interactions 342 Maternal diet modulated the age effects on offspring. Differences in survival, size, and prey 343 consumption between offspring of older and younger mothers were most pronounced when 344 mothers had restricted diets. Contrary to our second hypothesis and previous models 345 (Vijendravarma et al., 2010; Hafer et al., 2011; van den Heuvel et al., 2016), offspring from 346 older, diet-restricted mothers had the best overall performance. One possible explanation is 347 that the observed effects are anticipatory, where mothers prime their offspring against 348 resource limitation. Alternatively, it could be that our restricted diet treatment was not 349 sufficiently stressful to elicit phenotypic costs on offspring, and the mild dietary restriction 350 may have buffered negative maternal age effects (Gribble et al., 2014; Hibshman et al., 351 2016). Stronger dietary stress could reverse this pattern, which should be investigated in 352 future experiments. 353 The results of our study also highlight the context-dependence of maternal age effects, 354 consistent with evidence that outcomes can vary across environmental conditions and taxa 355 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 20 (Beckerman et al., 2006; Marshall et al., 2010; Kuijper & Johnstone, 2018; Zirbel & Alto, 356 2018). 357 358 4.3 Proximate mechanisms and ecological implications 359 The most important finding was that offspring of older mothers grew larger while consuming 360 fewer prey, which suggests an increased efficiency of energy conversion (Abrams et al., 361 1996; Arendt, 1997). Whether this reflects altered metabolism, reduced energy expenditure 362 (e.g., foraging activity), or other physiological mechanisms warrants further study. 363 Another possible explanation for reduced prey consumption between offspring of old 364 and young mothers could be the variation in their predation strategy. In arachnids, 365 superfluous killing—killing more prey than consumed—has been documented in both mites 366 and spiders (Metz et al., 1988; Maupin & Riechert, 2001; López‐Mercadal et al., 2024). 367 Offspring of younger mothers might engage more in superfluous killing whereas those of 368 older mothers might not. Although we found no direct evidence of partially consumed prey 369 eggs, this remains a hypothesis for future testing. 370 Reduced prey consumption by the later-produced offspring could provide adaptive 371 benefits. Later-produced offspring may encounter depleted resources because of utilisation by 372 mothers and earlier-produced offspring. Lower consumption requirements may reduce 373 intraspecific competition among siblings and with mothers, which enhances both maternal 374 and offspring survival. As a solitary predator, reduced food demand in the later-produced 375 A. herbicolus could also facilitate dispersal and colonisation of new patches. Maternal age 376 may thus act as a cue of environmental change, enabling offspring to anticipate resource 377 scarcity (Vargas et al., 2012) and the onset of environmental stresses (Rossi et al., 2016). 378 Furthermore, such age-related changes in maternal provisioning strategies may enhance long-379 term fitness by diversifying brood performance (Cameron et al., 2017). 380 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 21 381 4.4 Future directions 382 Our study was conducted in an asexual system, which avoids confounding paternal effects 383 but limits generalisation to sexually reproducing taxa (Castonguay & Angers, 2012; 384 Verhoeven & Preite, 2014). Moreover, prey consumption declined with maternal age in both 385 diet treatment groups. This dynamic could alter effective resource availability (i.e., more 386 leftovers from older individuals) and influence maternal provisioning, potentially 387 contributing to the observed inverse Lansing effect. Future work on sexually reproducing 388 species would help determine whether similar age-related patterns and maternal provisioning 389 dynamics occur under biparental reproduction. 390 The persistence of maternal-age effects across generations remains uncertain. Some 391 studies report only immediate maternal effects, while others document transgenerational 392 persistence (Plaistow et al., 2015; Goos et al., 2019; Wylde et al., 2019). Assessing whether 393 maternal-age effects extend beyond a single generation, and whether these effects are 394 additive or interactive with subsequent generations and environmental factors, can provide 395 important insights into non-genetic inheritance. 396 Lastly, offspring may respond differently to variation in age-related maternal 397 provisioning (Plaistow et al., 2015). Whether the observed inverse Lansing effect in 398 A. herbicolus is a compensatory strategy, and whether this leads to trade-offs in later life, 399 should be examined further. 400 401 5 Conclusions 402 In summary, we observed evidence of an inverse Lansing effect, where offspring of older 403 mothers performed better than those of younger mothers during development. Our study 404 highlights that maternal diet can affect offspring development in A. herbicolus, and that the 405 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted October 16, 2025. ; https://doi.org/10.1101/2025.10.16.682747doi: bioRxiv preprint 22 effect depends strongly on maternal age at oviposition. The interaction between maternal diet 406 and age suggests that maternal reproductive decisions and maternal effects are not static but 407 change over time, and that the timing of oviposition plays a crucial role in shaping offspring 408 outcomes. Maternal age may therefore play a key role in maintaining variation in life 409 histories and should be considered more explicitly when examining maternal effects and 410 offspring performance. 411 412

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