bioRxiv preprint doi: https://doi.org/10.1101/2021.03.11.434967; this version posted April 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Intergenerational Effects of Early Life Starvation on Life-History, Consumption, 2 and Transcriptome of a Holometabolous Insect 3 4 Sarah Catherine Paul1,a, Pragya Singh1,a, Alice B. Dennis2, Caroline Müller1* 5 6 1Chemical Ecology, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, 7 Germany 8 2 Evolutionary Biology & Systematic Zoology, University of Potsdam Karl-Liebknecht- 9 Strasse 24-25, 14476 Potsdam 10 a shared first authorship 11 *corresponding author: [email protected] 12 13 Mail addresses: SCP: [email protected], PS: 14 [email protected], ABD: [email protected], CM: 15 [email protected] 16 17 Running title: Intergenerational effects on sawfly 18 19 Keywords 20 Intergenerational effects, sawfly, starvation, transcriptome, parental effects, 21 compensatory growth. 22 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.11.434967; this version posted April 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 23 ABSTRACT: Intergenerational effects, also known as parental effects in which the 24 offspring phenotype is influenced by the parental phenotype, can occur in response 25 to parental early life food-limitation and adult reproductive environment. However, 26 little is known about how these parental life stage-specific environments interact with 27 each other and with the offspring environment to influence offspring phenotype, 28 particularly in organisms that realize distinct niches across ontogeny. We examined 29 the effects of parental early life starvation and adult reproductive environment on 30 offspring traits under matching or mismatching offspring early life starvation 31 conditions using the holometabolous, haplo-diploid insect Athalia rosae (turnip 32 sawfly). We show that the parental early life starvation treatment had context- 33 dependent intergenerational effects on the life-history and consumption traits of 34 offspring larvae, partly in interaction with offspring conditions and sex, while there 35 was no significant effect of parental adult reproductive environment. In addition, while 36 offspring larval starvation led to numerous gene- and pathway-level expression 37 differences, parental starvation impacted fewer genes and only the ribosomal 38 pathway. Our findings reveal that parental starvation evokes complex 39 intergenerational effects on offspring life-history traits, consumption patterns as well 40 as gene expression, although the effects are less pronounced than those of offspring 41 starvation. 42 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.11.434967; this version posted April 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 43 Introduction 44 Intergenerational effects, more commonly known as parental effects, are defined as 45 causal influences of parental phenotype on offspring phenotype that are likely 46 mediated by non-DNA sequence-based inheritance (Wolf and Wade 2009; Perez and 47 Lehner 2019). Together with transgenerational effects, which occur across several 48 generations, they play a key role in the ecology and evolution of organisms (Badyaev 49 and Uller 2009) and impact the responses of individuals to changing environmental 50 conditions (Sánchez‐Tójar et al. 2020). One important environmental factor that can 51 rapidly change during an organism’s lifetime and across generations is food 52 availability. Periods of starvation are commonly encountered by insects in the wild 53 (Jiang et al. 2019) and, when experienced early in life, often have long-lasting effects 54 on an individual’s phenotype, affecting life-history, consumption, and behavior 55 (Miyatake 2001; Wang et al. 2016; McCue et al. 2017). Intergenerational effects of 56 starvation events on offspring developmental trajectories have been found to differ 57 depending on the exact starvation regime, sex, and traits that are considered 58 (Saastamoinen et al. 2013; McCue et al. 2017; Paul et al. 2019). To fully elucidate 59 the adaptive nature of intergenerational effects, studies should encompass different 60 phases in development of the parental life cycle and how such life stage-specific 61 experience affects offspring phenotype (English and Barreaux 2020). 62 The early life environment can shape the phenotype of an individual and its 63 offspring via distinct routes (Monaghan 2008; Burton and Metcalfe 2014; Taborsky 64 2017). Individuals experiencing a benign environment in early life may pass on their 65 superior condition to their offspring (van de Pol et al. 2006), often called silver spoon 66 or carry-over effects (Monaghan 2008), resulting in offspring that are better able to 67 deal with stressful conditions (Franzke and Reinhold 2013). Similarly, negative 68 effects of a poor start in life might also be passed on, resulting in offspring less able 69 to cope with stressful conditions (Naguib et al. 2006). Alternatively, such stressed 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.11.434967; this version posted April 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 70 individuals may produce offspring that are buffered against stressors, for example, 71 through parental provisioning (Valtonen et al. 2012; Pilakouta et al. 2015; Hibshman 72 et al. 2016). Finally, identical environmental conditions experienced in a certain stage 73 (i.e. a match between parental and offspring environment) may be predicted to be 74 optimal for the offspring (match-mismatch scenario). Evidence for such predictive 75 adaptive effects has been found in several species (Raveh et al. 2016; Le Roy et al. 76 2017), but is weak in others (Uller et al. 2013). Each of these avenues of 77 intergenerational effects are not mutually exclusive. For example, offspring in 78 mismatched environments may do relatively better if their parents were of high 79 quality, due to silver spoon effects (Engqvist and Reinhold 2016). Fully reciprocal 80 designs are needed to determine the relative importance of the experience in a 81 certain life stage or generation on such developmental plasticity (Groothuis and 82 Taborsky 2015). 83 In addition, parental investment in offspring can vary depending on the condition of 84 the parent’s mate (Ratikainen and Kokko 2010). Both males and females have been 85 shown to enhance investment in response to perceived increases in mate quality or 86 attractiveness (Cunningham and Russell 2000; Cornwallis and O'Connor 2009). 87 Changes in parental investment based on mate cues may potentially counteract or 88 augment the effects of the parental early life environment on offspring phenotype. For 89 example, poor early life conditions can lead to poor quality of mates and therefore 90 affect mating success and reproductive investment (Hebets et al. 2008; Vega-Trejo et 91 al. 2016). As niches often shift across an individual’s lifetime, both parental early and 92 later life environments and their interaction must be considered. 93 With discrete phases during development, insects, particularly holometabolous 94 insects, present ideal organisms in which to investigate how intra- and 95 intergenerational environmental cues, such as food limitation and mate quality, might 96 influence individual phenotypes (English and Barreaux 2020). Periods of low 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.11.434967; this version posted April 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 97 resource availability during larval development are known to influence developmental 98 trajectories with knock-on effects on adult size, reproductive success and adult 99 starvation resistance (Boggs and Niitepõld 2016; Wang et al. 2016). Individuals may 100 respond to periods of starvation with increased food uptake (Regalado et al. 2017) as 101 well as compensatory growth (i.e. a faster growth rate) or catch-up growth (i.e. 102 attaining a minimum size by prolonging the larval development) (Hector and 103 Nakagawa 2012). Moreover, starvation leads to substantial shifts in gene expression 104 within individuals (Moskalev et al. 2015; Jiang et al. 2019; Etebari et al. 2020; 105 Farahani et al. 2020). However, little is known about how long these effects may 106 persist (McCue et al. 2017) and to what extent parental starvation may affect gene 107 expression of offspring experiencing similar or dissimilar conditions. 108 In the present study, we investigated the effects of parental early life starvation 109 and parental reproductive environment on offspring (i.e. intergenerational effects), 110 under matching or mismatching larval starvation conditions, using the turnip sawfly, 111 Athalia rosae (Hymenoptera: Tenthredinidae). Adult females oviposit on various 112 species of Brassicaceae, including crop species, on which A. rosae can become a 113 pest (Riggert 1939; Sawa et al. 1989). The leaf-consuming larvae can