• Energy Research

Climate change can severely impact species that depend on temporary resources by inducing phenological mismatches between consumer and resource seasonal timing. In the winter moth, warmer winters caused eggs to hatch before their food source, young oak leaves, became available. This phenological mismatch changed the selection on the temperature sensitivity of egg development rate. However, we know little about the fine-scale fitness consequences of phenological mismatch at the individual level and how this mismatch affects population dynamics in the winter moth. To determine the fitness consequences of mistimed egg hatching relative to timing of oak budburst, we quantified survival and pupation weight in a feeding experiment. We found that mismatch greatly increased mortality rates of freshly hatched caterpillars, as well as affecting caterpillar growth and development time. We then investigated whether these individual fitness consequences have population-level impacts by estimating the effect of phenological mismatch on population dynamics, using our long-term data (1994–2021) on relative winter moth population densities at four locations in The Netherlands. We found a significant effect of mismatch on population density with higher population growth rates in years with a smaller phenological mismatch. Our results indicate that climate change-induced phenological mismatch can incur severe individual fitness consequences that can impact population density in the wild.

Field data on winter moths were collected yearly since 1994 in four forests around Arnhem, the Netherlands, using simple funnel traps to catch adult moths in winter (see [Van Asch et al. 2013, Nat Clim Change] for details). Eggs collected from these wild adults were kept in a field shed at the Netherlands Institute of Ecology. Deposited field data for the period 1994–2021 include per year: number of adult moths collected, with for each moth (individual-based data with individual identifier): number of eggs laid, spring seasonal timing of their eggs kept in our field shed, and spring seasonal timing of budburst of oak trees in the field on which adults were caught. Experimental data were collected in a caterpillar feeding experiment in the Spring of 2021, using eggs from the long-term field monitoring (described above). The experiment consisted of a split-brood design, where the timing of hatching of eggs laid by each female was manipulated to induce staggered hatching. Caterpillars were then divided over different photoperiod treatments (constant photoperiod or naturally changing photoperiod) and different phenological mismatch treatments (hatching before [0–4 days] or after oak budburst [1–5 days], and then fed with oak leaves accordingly). Deposited experimental data include per caterpillar (individual-based data with individual identifier): parent origin (Catch area, tree, and date), hatch date, death date (if died before pupating), pupation date, pupation weight, date of adult emerging, adult weight, and adult sex. Climate change can severely impact species that depend on temporary resources by inducing phenological mismatches between consumer and resource seasonal timing. In the winter moth, warmer winters caused eggs to hatch before their food source, young oak leaves, became available. This phenological mismatch changed the selection on the temperature sensitivity of egg development rate. However, we know little about the fine-scale fitness consequences of phenological mismatch at the individual level and how this mismatch affects population dynamics in the winter moth. To determine the fitness consequences of mistimed egg hatching relative to timing of oak budburst, we quantified survival and pupation weight in a feeding experiment. We found that mismatch greatly increased mortality rates of freshly hatched caterpillars, as well as affecting caterpillar growth and development time. We then investigated whether these individual fitness consequences have population-level impacts by estimating the effect of phenological mismatch on population dynamics, using our long-term data (1994–2021) on relative winter moth population densities at four locations in the Netherlands. We found a significant effect of mismatch on population density with higher population growth rates in years with a smaller phenological mismatch. Our results indicate that climate change-induced phenological mismatch can incur severe individual fitness consequences that can impact population density in the wild. Funding provided by: Rijksuniversiteit GroningenCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001721Award Number: IVA AL 3.2C DIS

ABSTRACT Climate change is rapidly altering the environment and many species will need to genetically adapt their seasonal timing to keep up with these changes. Insect development rate is largely influenced by temperature, but we know little about the mechanisms underlying the temperature sensitivity of development. Here, we investigate seasonal timing of egg hatching in the winter moth, one of the few species which has been found to genetically adapt to climate change, likely through selection on temperature sensitivity of egg development rate. To study when during development winter moth embryos are most sensitive to changes in ambient temperature, we gave eggs an increase or decrease in temperature at different moments during their development. We measured their developmental progression and time of egg hatching, and used fluorescence microscopy to construct a timeline of embryonic development for the winter moth. We found that egg development rate responded more strongly to temperature once embryos were in the fully extended germband stage. This is the phylotypic stage at which all insect embryos have developed a rudimentary nervous system. Furthermore, at this stage, timing of ecdysone signaling determines developmental progression, which could act as an environment dependent gateway. Intriguingly, this may suggest that, from the phylotypic stage onward, insect embryos can start to integrate internal and environmental stimuli to actively regulate important developmental processes. As we found evidence that there is genetic variation for temperature sensitivity of egg development rate in our study population, such regulation could be a target of selection imposed by climate change.

AbstractTo accurately predict species’ phenology under climate change, we need to gain a detailed mechanistic understanding of how different environmental cues interact to produce the seasonal timing response. In the winter moth (Operophtera brumata), seasonal timing of egg hatching is strongly affected by ambient temperature and has been under strong climate change-induced selection over the past 25 years. However, it is unclear whether photoperiod received at the egg stage also influences timing of egg hatching. Here, we investigated the relative contribution of photoperiod and temperature in regulating winter moth egg development using two split-brood experiments. We experimentally shifted the photoperiod eggs received by 2–4 weeks compared to the actual calendar date and measured the timing of egg hatching, both at a constant temperature and in combination with two naturally changing temperature treatments – mimicking a cold and a warm year. We found an eight-fold larger effect of temperature compared to photoperiod on egg development time. Moreover, the very small photoperiod effects we found were outweighed by both between- and within-clutch variation in egg development time. Thus, we conclude that photoperiod received at the egg stage does likely not play a substantial role in regulating the seasonal timing of egg hatching in the winter moth. These insights into the regulatory mechanism of seasonal timing could have important implications for predicting insect climate change adaptation, as we might expect different targets of selection depending on the relative contribution of different environmental cues.

The ecological impact of artificial light at night (ALAN) is an increasingly recognized process that accompanies expanding urbanization. Yet, we have limited knowledge on the impact of ALAN on wild species, and on the potential to mitigate any negative effects by using different light sources and colors. In birds, effects of ALAN on activity levels are reported for several species and, hence, their daily energy expenditure (DEE) may be affected. DEE is a potent mediator of life-history trade-offs and fitness and thus an important aspect to consider when examining the potential long-term ecological effects of ALAN. Previous work has suggested that birds exposed to ALAN show higher levelsof provisioning and nocturnal activity, suggesting that white ALAN increases DEE. Other factors regulating DEE, such as provisioning behavior and food availability, might also respond to ALAN and thus indirectly affect DEE. We tested the hypothesis that ALAN increases DEE using an experimental setup where four previously unlit transects were illuminated with either white, green, or red LED light, or left dark as a control treatment.This setup was replicated in eight locations across the Netherlands.Wemeasured DEE of our focal species, the great tit (Parusmajor), using a novel doubly labeled water technique that uses breath rather than blood samples. Contrary to our expectations, birds feeding their offspring under white and green ALAN showed lower DEE compared to birds in the control dark treatment. Differences in chick provisioning activity did not explain this result, as neither visit rates nor daily activity timing was affected by light treatment. However, food availability under white and green light was much higher compared to red light and the dark control. This difference strongly suggests that the lower DEE under white andgreen ALAN sites is a consequence of higher food availability in these treatments. This result shows that there can be positive, indirect effects of ALAN for breeding song birds which may balance against the negative direct effects shown in previous studies.

To accurately predict species' phenology under climate change, we need to gain a detailed mechanistic understanding of how different environmental cues interact to produce the seasonal timing response. In the winter moth (Operophtera brumata), seasonal timing of egg hatching is strongly affected by ambient temperature and has been under strong climate change-induced selection over the past 25 years. However, it is unclear whether photoperiod received at the egg stage also influences timing of egg hatching. Here, we investigated the relative contribution of photoperiod and temperature in regulating winter moth egg development using two split-brood experiments. We experimentally shifted the photoperiod eggs received by 2-4 weeks compared to the actual calendar date and measured the timing of egg hatching, both at a constant temperature and in combination with two naturally changing temperature treatments – mimicking a cold and a warm year. We found an eight-fold larger effect of temperature compared to photoperiod on egg development time. Moreover, the very small photoperiod effects we found were outweighed by both between- and within-clutch variation in egg development time. Thus, we conclude that photoperiod received at the egg stage does likely not play a substantial role in regulating the seasonal timing of egg hatching in the winter moth. These insights into the regulatory mechanism of seasonal timing could have important implications for predicting insect climate change adaptation, as we might expect different targets of selection depending on the relative contribution of different environmental cues. We investigated whether photoperiod received at the egg stage influences the seasonal timing of egg hatching in the winter moth, both as a cue on its own and in interaction with temperature. In two split-brood experiments, we determined egg hatching date after giving eggs either an early or late season photoperiod treatment, with naturally changing day lengths shifted 2-4 weeks earlier or later compared to the actual calendar date. Temperature was kept constant in the first experiment, while the second experiment also incorporated two naturally changing temperature treatments – mimicking a cold and a warm year – to investigate the relative contribution of temperature and photoperiod. Eggs were collected from wild females (31 clutches and 20 clutches, for experiment 1 and 2 respectively), with each clutch of eggs split into sub-clutches and divided over the different photoperiod and photoperiod-temperature treatments. Raw egg hatching data (i.e. 2-3 observations per week of the number of freshly hatched caterpillars per sub-clutch) have been processed by calculating the median hatching date per sub-clutch (D50, i.e. the day at which 50% of the sub-clutch has hatched). D50 data were used for the statistical analyses and have been deposited here, together with the raw temperature logger data from the experiment.

# Temperature has an overriding role compared to photoperiod in regulating the seasonal timing of winter moth egg hatching ## Description of the data and file structure Raw data belonging to the manuscript "Temperature has an overriding role compared to photoperiod in regulating the seasonal timing of winter moth egg hatching". We investigated whether photoperiod received at the egg stage influences the seasonal timing of egg hatching in the winter moth, both as a cue on its own and in interaction with temperature. In two split-brood experiments, we determined egg hatching date after giving eggs either an early or late season photoperiod treatment, with naturally changing day lengths shifted 2-4 weeks earlier or later compared to the actual calendar date. Temperature was kept constant in the first experiment, while the second experiment also incorporated two naturally changing temperature treatments – mimicking a cold and a warm year – to investigate the relative contribution of temperature and photoperiod. **NB: Treatment names consist of photoperiod treatment: (1) control [0 weeks shift], (2) very early season photoperiod [-4 weeks], (3) early season photoperiod [-2 weeks/K], (4) late season photoperiod [+2 weeks/L], and (5) very late season photoperiod [+4 weeks]; and temperature treatment: mimicking cold year 1999 [K] and warm year [W]** \--- Split-brood experiment 1: Photoperiod experiment 2013-2014 files \ Contains all experimental data collected from the photoperiod experiment (EggPhot2014), incl. for each sub-clutch (ClutchID) their origin (TubeID=female parent - 31 females in total - incl. catch info for this female parent i.e. Area, Site, Tree and NovemberDate=Catch date as julian dates with origin 2013-10-31); the treatment the ClutchID was assigned to (Treatment); the number of eggs in the sub-clutch (Eggs, at least 12); and when the sub-clutch hatched (D50Calc, i.e. julian dates with origin 2014-03-31). **NB: Median hatching date (D50Calc) per sub-clutch was only calculated if at least 10 eggs in the sub-clutch hatched.** \ Specifies the photoperiod treatment assignment for each ventilated box that eggs were kept in during the experiment. Needed to interpret the temperature data from the temperature loggers (see below). In total, 3 boxes per treatment (3x5 treatments = 15 boxes). \ Temperature logger data for boxes 1-5. \ Temperature logger data for boxes 6-10. \ Temperature logger data for boxes 11-15. Logger data per logger (LoggerName) measuring the Temperature for each ventilated box(LocationName/Location) for each measuring timepoint (Year, Month, Day, Date, Hour, Minute, Time), as obtained from the internal winter moth Access database (SysUser=who entered the data in the database, SysDate=when data entry happened, TempID and LocationID=database specific IDs). \--- Split-brood experiment 2: Photoperiod-temperature experiment 2000-2001 files \ Contains all experimental data collected from the photoperiod experiment (EggPhotTemp2001, ExperimentName=RN2001), with the same variables as described for above for . Treatment [KK]=Cold year and -2 weeks photoperiod; Treatment [WL]=Warm year and +2 weeks photoperiod. \ Temperature logger data for used incubators (indicated by Treatment name), with the same variables as described for above for . \--- Experimental photoperiod and temperature treatments \ Photoperiod treatments as used in the Photoperiod experiment (1 tab per treatment), giving the sunrise and sunset times in hours (HH:MM) for the calendar day the treatment was shifted to (date) for every day of the experiment (actual date). Note that this experiment started on January 15, while the same [-2 weeks] and [+2 weeks] treatments were used in the Photoperiod-temperature experiment, but starting earlier on December 12. \ \ Temperature treatments mimicked a specific year (Cold: 1973, Warm: 1999). For each day, a three-phase temperature cycle was used: 6 hours at the daily minimum temperature (h1), 12 hours at the mean of the daily maximum and the daily average (h7), and 6 hours at the daily average for each year (h19). ## Code/Software All code needed to reproduce the manuscript's data analysis using R are included.See the file for used R version and package versions. \--- Split-brood experiment 1 Script: <1_EggPhotoExp2014_analysis.R> R script to reproduce the analysis and visualization (incl. manuscript figures) of the 2014 winter moth egg photoperiod experiment: Does photoperiod received at the egg stage affect the seasonal timing of egg hatching? \--- Split-brood experiment 2 Script: <2_scripts/2_EggPhotoTempExp2001_analysis.R> R script to reproduce the analysis and visualization (incl. manuscript figures) of the 2001 winter moth egg photoperiod-temperature experiment: What is the relative contribution of photoperiod and temperature on the timing of egg hatching? \--- Visualization of experimental photoperiod and temperature treatments Script: <3_visualize-treatments.R> R script to visualize the photoperiod and temperature treatments as used in the two experiments, showing how they change over the season to mimic naturally changing environmental conditions. See for used R version and package versions. To accurately predict species’ phenology under climate change, we need to gain a detailed mechanistic understanding of how different environmental cues interact to produce the seasonal timing response. In the winter moth (Operophtera brumata), seasonal timing of egg hatching is strongly affected by ambient temperature and has been under strong climate change-induced selection over the past 25 years. However, it is unclear whether photoperiod received at the egg stage also influences timing of egg hatching. Here, we investigated the relative contribution of photoperiod and temperature in regulating winter moth egg development using two split-brood experiments. We experimentally shifted the photoperiod eggs received by 2-4 weeks compared to the actual calendar date and measured the timing of egg hatching, both at a constant temperature and in combination with two naturally changing temperature treatments – mimicking a cold and a warm year. We found an eight-fold larger effect of temperature compared to photoperiod on egg development time. Moreover, the very small photoperiod effects we found were outweighed by both between- and within-clutch variation in egg development time. Thus, we conclude that photoperiod received at the egg stage does likely not play a substantial role in regulating the seasonal timing of egg hatching in the winter moth. These insights into the regulatory mechanism of seasonal timing could have important implications for predicting insect climate change adaptation, as we might expect different targets of selection depending on the relative contribution of different environmental cues. We investigated whether photoperiod received at the egg stage influences the seasonal timing of egg hatching in the winter moth, both as a cue on its own and in interaction with temperature. In two split-brood experiments, we determined egg hatching date after giving eggs either an early or late season photoperiod treatment, with naturally changing day lengths shifted 2-4 weeks earlier or later compared to the actual calendar date. Temperature was kept constant in the first experiment, while the second experiment also incorporated two naturally changing temperature treatments – mimicking a cold and a warm year – to investigate the relative contribution of temperature and photoperiod. Eggs were collected from wild females (31 clutches and 20 clutches, for experiment 1 and 2 respectively), with each clutch of eggs split into sub-clutches and divided over the different photoperiod and photoperiod-temperature treatments. Raw egg hatching data (i.e. 2-3 observations per week of the number of freshly hatched caterpillars per sub-clutch) have been processed by calculating the median hatching date per sub-clutch (D50, i.e. the day at which 50% of the sub-clutch has hatched). D50 data were used for the statistical analyses and have been deposited here, together with the raw temperature logger data from the experiment.