DOI: 10.5061/dryad.nk98sf7ss
title: Alternative developmental and transcriptomic responses to host plant water limitation in a butterfly metapopulation

description: The dataset is from a study examining the effects of host plant water
 stress on the developmental and transcriptomic responses of its specialist
 Lepidopteran herbivore. The study combines host plant metabolic profiling
 with development assays and full-transcriptome sequencing of herbivore
 larvae. First, we profiled metabolic differences between well-watered and
 water-limited ribwort plantain (Plantago lanceolata) using proton nuclear
 magnetic resonance spectroscopy (1H-NMR). Second, we tested how
 performance of developing Glanville fritillary (Melitaea cinxia) larvae
 was affected by host plant water limitation experienced at different
 larval developmental stages. Third, we examined larval gene regulatory
 responses to water limited host plants by sequencing full transcriptomes
 of 77 female larvae (RNA seq). Finally, to examine intrapopulation
 variation in the responses of the larvae, we compared the phenotypic and
 transcriptomic responses across full-sib families originating from
 different parts of the metapopulation. In this dataset, we provide data
 for the P. lanceolata metabolite responses to water limitation and
 developmental responses of the M. cinxia larvae to feeding on water
 limited P. lanceolata. The transcriptomic data are available from
 NCBI's Gene Expression Omnibus, with the accession number GSE159376.
description: We collected seeds from a natural population in the Åland Islands
 (60.196° Lat., 20.704° Lon.) and after germination planted 360 plants in
 0.75 litre pots (two saplings each). We reared the plants for three months
 in controlled greenhouse conditions (ca. 40 ml water / pot daily, 15L:9D
 photoperiod with 26:18 °C temperature cycle) before initiating the water
 limitation treatment. We exposed 240 plants to a water limitation
 treatment in which daily watering was reduced by 50% compared to controls
 (20ml per pot).<br> <br> Each morning prior to watering (9-10
 am), we randomly harvested <em>P. lanceolata</em> leaves from
 control and water limited plants and cut them into 2.25cm2 pieces,
 discarding the basal and tip parts of the leaves. We used these pieces to
 feed larvae during the experiment (see below), and selected a random
 subset of six pieces from both treatments for metabolomics assays. For the
 assays, we recorded the fresh biomass of each piece of leaf, snap froze
 the pools in liquid nitrogen and stored them in -80 °C. We then freeze
 dried the samples for 48 hours after which we measured their dry weight,
 estimated relative water content in the sample [i.e. (fresh mass – dry
 mass) / fresh mass], ground the dried pool samples in dry ice using a
 sterile pestle and prepared the finely ground tissue powder for proton
 nuclear magnetic resonance spectrometry (1H-NMR) following the protocol
 described by Kim et al. (2010). The 1H-NMR spectra of the <em>P.
 lanceolata</em> pool samples was recorded at the Finnish Biological
 NMR Center (Institute of Biotechnology, University of Helsinki,
 http://www.biocenter.helsinki.fi/bi/) using a Bruker Avance III HD NMR
 spectrometer (Bruker BioSpin, Germany) operated at 1H frequency of 850.4
 MHz equipped with a cryogenic probe head. The 1H-NMR spectra were aquired
 at a 25 °C temperature with a “zgpr” pulse sequence.
 <br> After obtaining the spectra, we corrected the
 baseline, used automatic peak detection, binned the observed peaks into
 0.04 ppm bins for all pool samples and normalized them according to the
 TSP signal variation and dry mass of the sample using MNOVA v.10.0.2
 software (Mestrelab research S.L., Spain). We further annotated the peaks
 within the binned intervals based on signals of pure compounds for
 Aucubin, Catalpol and Verbascoside or from published characteristic
 signals (Agudelo-Romero et al., 2014; Gogna et al., 2015; Kim et al.,
 2010). <em>M. cinxia</em> larval
 families, treatments and developmental assays
 We created experimental full-sib larval family groups by mating
 individuals originating from local populations across the natural Åland
 islands metapopulation. From nine mated females, we picked two larval
 groups of a minimum of eighty larvae each to enter the experiment. On the
 day after hatching, we divided the larval groups into four smaller groups
 of twenty larvae each and placed them on separate petri dishes (9 cm
 diameter, 1.5 cm deep) lined with filter paper. We then randomly assigned
 each of the dishes to one of four different treatments mimicking different
 temporal exposures to drought stressed host plants. In addition to a
 control treatment, in which we fed the larvae with control reared host
 plants only, the larvae experienced water limited host plant at different
 stages during pre-diapause development: (1) at late pre-diapause
 development during 3rd and 4th larval instars, (2) at early pre-diapause
 development during 1st and 2nd larval instars, and (3) throughout their
 pre-diapause development from the 1st to the 5th larval instar. In all
 treatments, we fed the larvae daily with pieces of host plant leaf tissue
 corresponding to the treatment. We provided <em>ad
 libitum</em> food such that – in order to avoid feeding on old leaf
 tissue with potentially altered phytochemistry – the larvae consumed most
 but not all of the leaf tissue during the next 24 hours after
 provisioning. During the experiment, we recorded
 development time, body mass at diapause and mortality during development.
 Once the last larva on a petri dish had entered diapause, we allowed them
 to spend another four days in normal rearing temperature and photoperiod,
 after which we measured their body mass and placed them in climate
 chambers (+5 °C, 95% air humidity) for diapause. We allowed the larvae to
 diapause for six months, after which we woke them up and recorded
 overwintering mortality.
description: The data are accompanied by a README.txt file describing all data
 fields in all data files. Note that although larval
 development time and body mass related data is provided at the individual
 level, the records do not have individual level IDs, Therefore the
 development time and diapause body mass data cannot be combined with each
 other to examine their association within each individual. This is because
 the larvae spend their pre-diapause development and overwintering in
 full-sib larval groups making it very challenging to track individual
 larvae across their development.<br> <br> Note also that the
 data also contains some outlier cases we suggest removing before any
 analyses. Larvae in the control treatment of the first replicate larval
 group of family F-5 (clutch ID 17) had a mortality of 55%. As the larvae
 in this group were developing poorly in general, we concluded it to be an
 outlier case, potentially suffering from a disease or some other unknown
 agent. For transparency we provide these cases with the data, but
 recommend excluding this larval group from any further analyses. In all
 data files regarding <em>M. cinxia</em> larvae, records for
 this group have been marked as outlier cases with an "x" in the
 data column "outlier".