DOI: 10.5061/dryad.tmpg4f553
title: Data from: What goes in must come out? The metabolic profile of plants and caterpillars, frass, and adults of Asota (Erebidae: Aganainae) feeding on Ficus (Moraceae) in New Guinea

description: Insect herbivores have evolved a broad spectrum of adaptations in response
 to the diversity of chemical defences employed  by plants. Here
 we focus on two species of New Guinean Asota and determine how these
 specialist moths deal with the leaf alkaloids of their fig (Ficus) hosts.
 As each focal Asota species is restricted to one of three chemically
 distinct species of Ficus, we also test whether these specialized
 interactions lead to similar alkaloid profiles in both Asota species. We
 reared Asota caterpillars on their respective Ficus hosts in natural
 conditions and analyzed the alkaloid profiles of leaf, frass, caterpillar,
 and adult moth samples using UHPLC–MS/MS analyses. We identified 43
 alkaloids in our samples. Leaf alkaloids showed various fates. Some were
 excreted in frass or found in caterpillars and adult moths. We also found
 two apparently novel indole alkaloids likely synthesized de novo by the
 moths or their microbiota—in both caterpillar and adult tissue
 but  not in leaves or frass. Overall, alkaloids unique or largely
 restricted to insect tissue were shared across moth species despite
 feeding on different hosts. This indicates that a limited number of plant
 compounds have a direct ecological function that is conserved among the
 studied species. Our results provide evidence for the importance of
 phytochemistry and metabolic strategies in the formation of plant–insect
 interactions and food webs in general. Furthermore, we provide a new
 potential example of insects acquiring chemicals for their benefit in an
 ecologically relevant insect genus.
description: Please refer to Fontanilla et al. (2022) for detailed methods.
 These data were collected from plant and insect tissue in New Guinea at
 the Binatang Research Centre and analysed using UHPLC-MS/MS at the
 University of Turku.
description: # What Goes in Must Come Out? The Metabolic Profile of Plants and
 Caterpillars, Frass, And Adults of Asota (Erebidae: Aganainae) Feeding on
 Ficus (Moraceae) in New Guinea
 [https://doi.org/10.5061/dryad.tmpg4f553](https://doi.org/10.5061/dryad.tmpg4f553) These data comprise 13 files and one annotated R script entitled 'Asota MS Analysis R 250923'. These data report the alkaloidal compounds extracted from Asota moths (Erebidae: Aganainae). We studied two species of Asota (A. eusemioides and A. heliconia) across three species of Ficus (F. septica, F. pachyrrhachis, and F. hispidioides) in a rearing experiment outlined in detail in Fontanilla et al. (2022). We collected moths at different developmental stages (caterpillars and adults) as well as their frass and leaves from their host plants. All samples were analysed using UHPLC-MS/MS and in total we found 43 alkaloidal compounds and using multivariate data analysis we explored the occurrence of compounds across sample types (including across adult body parts). Univariate analyses were used to explore compound occurrence based on trends discovered in these exploratory analyses. Below we report the experimental details needed to interpret these data. The first part of our experiment was aimed at rearing caterpillars on each host to adulthood; this allowed accurate identification of species and developmental stage, and confirmation that body size increased predictably across instars (Dyar 1890). We selected two individual trees from each host species (n = 6) and attempted to rear five individual first instar caterpillars (collected from the appropriate host species in the surrounding forest) to adulthood (n = 30). Individual caterpillars were placed on a single branch of the Ficus species from which they were sampled and enclosed in a fine breathable mesh for rearing. Larvae were reared on young, fully expanded, and healthy leaves until their last instar, and frass was regularly removed (at least daily) to keep conditions as clean as possible. Caterpillars were followed from 13/02/2018 until 01/03/18, by which time 21 out of 30 larvae had entered pupation. Eleven larvae were reared to adulthood and stored at − 20 °C before being freeze dried. Caterpillar body length was measured to the nearest 0.1 cm on a daily basis. We also collected 10 leaves from a separate branch of the same tree before placing the caterpillars (n = 48; leaves from one tree were excluded and one tree was sampled for eight leaves). These leaves were placed in an ice box and transferred to a − 20 °C freezer before being freeze dried at the Binatang Research Centre. For our main experiment selected 12 trees of each host species. For each tree, we aimed to place 15 individual caterpillars of the appropriate Asota species on individual branches covered by mesh bags. Due to the local availability of first instar caterpillars, however, we ended up with 29 caterpillars on F. septica, 32 on F. hispidioides, and 31 on F. pachyrrhachis, for a total of 92 caterpillars. As with the first part, caterpillars were allowed to feed only on young and healthy leaves.Once caterpillars had molted into the last instar, ca. one half (n = 44) were freeze dried and collected as larval samples while the other half (n = 48) were reared to adults. The former were starved for six hours prior to freeze drying to ensure that there was no contamination from plant metabolites. Caterpillars in the rearing treatment were allowed to feed for 24 hr after their final molt before being placed into pots with fresh leaves. Their frass was collected every 45 min for four and a half hours. The caterpillars were then returned to their host plants to pupate naturally, and adult moths were freeze dried after emergence. A total of 17 caterpillars from the rearing treatment died in the last instar and were removed; 15 of the 17 deaths were due to parasitoid attack. We also removed four caterpillars and one adult (and their frass samples) due to possible degradation, to give a total of 70 insect samples. Our aim was to collect six frass samples per caterpillar, for a total of 30 frass samples per tree. Because some samples were pooled in the chemical analysis due to low mass, we ended up with 170 frass samples instead of 180. In these cases, samples from the same individual—but not across individuals—were pooled. Frass was frozen immediately upon collection, as were the leaves upon which caterpillars had fed, and were later freeze dried. As the leaves on which the caterpillars fed were in generally poor condition and potentially influenced by caterpillar feeding itself, we again collected and freeze dried 10 leaves from a separate branch of the same tree to serve as baseline comparison.After accounting for excluded specimens, a total of 362 samples were used for subsequent analyses. All materials were sent to the University of Turku for chemical analyses. <br> ## Description of the data and file structure Our data consists of 13 files, each file is used in the analyses outlined in the paper and in the R script 'Asota MS Analysis R 250923'. Here we provide a summary of each file but link to sections of the R script for clarity. Asota Data 230720_ALKALOIDS_DEADCATS_070222_Means_NO_DEADCATS_2_Comm.txt =&gt; Community data (compound data by sample) used in multivariate analyses (## 07 PCA, pPCA and pCCA based on tissue type ####) Asota Data 230720_ALKALOIDS_DEADCATS_070222_Means_NO_DEADCATS_2_Env.txt =&gt; Environmental data (moth species, host species and sample type) used in multivariate analyses (## 07 PCA, pPCA and pCCA based on tissue type ####) Asota Data 230720_ALKALOIDS_DEADCATS_070222_Means_NO_DEADCATS_2_Individuals.txt =&gt; Individual data (a list that links individual moths to samples, e.g., a moth can be analysed as an adult for three body parts or as a caterpillar producing separate frass samples) used in multivariate analyses (## 07 PCA, pPCA and pCCA based on tissue type ####) Asota Data 230720_ALKALOIDS_DEADCATS_070222_Means_NO_DEADCATS_Adults_Only_2_Comm.txt =&gt; Community data (compound data by adult body part) used in multivariate analyses (## 09 PCA, pPCA and pCCA based on adult body parts ####) Asota Data 230720_ALKALOIDS_DEADCATS_070222_Means_NO_DEADCATS_Adults_Only_2_Env.txt =&gt; Environmental data (moth species, host species and adult body part) used in multivariate analyses (## 09 PCA, pPCA and pCCA based on adult body parts ####) Asota Data 230720_ALKALOIDS_DEADCATS_070222_Means_NO_DEADCATS_Adults_Only_2_Individuals.txt =&gt; Individual data (a list that links individual moths to samples, e.g., a moth can be analysed as an adult for three body parts) used in multivariate analyses (## 09 PCA, pPCA and pCCA based on adult body parts ####) Asota Data 230720_ALKALOIDS_DEADCATS_070222_Means_NO_DEADCATS_Singletons_2_Comm.txt =&gt; Community data (compound data by sample) used in multivariate analyses using means per individual (## 08 PCA and CCA based on tissue type with no repeated measures across samples ####) Asota Data 230720_ALKALOIDS_DEADCATS_070222_Means_NO_DEADCATS_Singletons_2_Env.txt =&gt; Environmental data (moth species, host species and sample type) used in multivariate analyses using means per individual (## 08 PCA and CCA based on tissue type with no repeated measures across samples ####) Asota Data 230720_ALKALOIDS_DEADCATS_NO_DEADCATS_080222_2.txt =&gt; Data used for univariate analyses of alkaloid occurence across tissue types (05 Zero inflated linear mixed model DEADCATS 070222) Asota Data 230720_ALKALOIDS_DEADCATS_NO_DEADCATS_080222_Adults_Only_2.txt =&gt; Data used for univariate analyses of alkaloid occurence across adult body parts (06 Zero inflated linear mixed model adults only DEADCATS 070222) Asota Data 230720_ALKALOIDS_DEADCATS_NO_DEADCATS_080222_Adults_Only_Reduced_2.txt =&gt;Data including only 'compounds of interest for adult body parts (03 Box plot for Adult Body Parts) Instar Data.txt =&gt; Data used for linear mixed models to assess size across larval instars (## 02 Instar Analysis ####) Instar Data_Asota_eusemioides.txt =&gt; Data used to test for a difference in larval development of Asota eusemoides across F. hispidioides and F. pachyrrhachis (## 02 Instar Analysis ####) ## Sharing/Access information These data were collected for the Fontanilla et al. (2022) and are not published elsewhere. ## Code/Software We have provided an annotated and reproducible R script in the file 'Asota MS Analysis R 250923' deposited on Zenodo.