EVOLUTION OF CYANIDE METABOLIMS IN APOSEMATIC BUTTERFLIES: from gene characterization to community ecology

Project Information

Cyanide Evolution

Grant agreement ID: 841230

DOI

10.3030/841230

Project closed

EC signature date 29 April 2019

Start date 1 January 2020

End date 2 September 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 224 933,76

EU contribution

€ 224 933,76

Coordinated by

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
United Kingdom

Periodic Reporting for period 1 - Cyanide Evolution (EVOLUTION OF CYANIDE METABOLIMS IN APOSEMATIC BUTTERFLIES: from gene characterization to community ecology)

Reporting period: 2020-01-01 to 2021-12-31

Summary of the context and overall objectives of the project

Heliconius butterflies has been studied by evolutionary biologists for over 150 years: they were used to exemplify the theory of mimicry as well as plant-insect coevolution. Yet, although the toxicity of Heliconius butterflies plays a central role in their macroevolution, the genetic and phenotypic bases of their chemical defences has been under studied. This project aimed to unravel how Heliconius butterflies evolved to be toxic: from characterizing the genes associate with this process to investigating how butterfly toxicity varies within and between species, in regard to their hostplants. This is important for society because while some generalist heliconiines species have become agricultural pest in passionfruit fields, at least one specialized species (H. naterreri) is vulnerable to extinction. Understanding the chemical mediation of the relationship between toxic butterflies and their food plants can be useful for conservation initiatives as well as the development of sustainable strategies for control of crop pests. Moreover, most information we have about these butterflies focus on communities in the Amazon Forest, but there is much less information about these butterflies in other ecosystems. This project also aimed to collect data on the distribution of heliconiine butterflies and their Passiflora hostplants across a 3,000km latitudinal gradient within Brazilian Atlantic Forest.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In WP1, we evaluated the fitness cost of toxicity in response to different larval diets and adult diets. We found that the balance between biosynthesis and sequestration in Heliconius is caused by biochemical plasticity in response to diet: Butterflies sequester their toxins from Passiflora when these compounds are available and biosynthesize them when they are absent in their hostplants. This biochemical plasticity has no fitness cost for a generalist Heliconius species which lay eggs in several hostplants (H. cydno), but is costly for a specialist species (H. melpomene). Moreover, we found that female Heliconius butterflies need pollen to keep their chemical defences, body weight and fertility during adulthood, while males are less dependent on their adult diet. Finally, we demonstrated with pedigree experiments that genetics only explains 15% of the variation in biosynthesized toxicity in H. erato and that females contribute more to the toxic profile of their progeny than males. These studies improved our knowledge on the chemical mediation of harmonic and disharmonic interactions between butterflies and their foodplants. It also showed that both genetics and phenotypic plasticity determine the toxic profile of Heliconius species (2 published papers, 1 submitted and 1 in prep)

In WP2, we were part of a study that created a genomic dataset of 58 heliconiine species, including a lot of genera and clades that were previously underrepresented in terms of genomic resources. This allowed us to understand how gene-flow, transposable elements, contraction-expansion of specific gene families, and gene networks has contributed for the diversification of heliconiines. We are currently using these dataset to establish the evolutionary trajectory of the genes involved in the biosynthesis of toxins in heliconiine butterflies. We found that CYP405As are duplicated in Heliconius and Eueides species, but not on other heliconiines. We used the gene-editing technology CRISPR-Cas and target-metabolomics to characterize the function of these genes (1 submitted paper and 1 in prep).

In WP3 we collected 272 Heliconiinae butterflies and 240 Passiflora plants across a 3,000 km latitudinal gradient within the Brazilian Atlantic Forest. Additionally, we also collected 137 Ithomiini butterflies during this fieldwork for a project in collaboration with Sanger Institute. Due to lockdowns and travel restrictions to control the spread of COVID-19m this project was delayed, but target-metabolomic analyses are being conducted on the collected samples which will allow us to understand how toxicity varies within and between species of heliconiine butterflies and how this is affected by their local Passiflora community (ongoing analyses - at least 2 publications are expected from this dataset).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

In this multidisciplinary project, we combined genomics, transcriptomics, target-metabolomics, the gene-Editing technology CRISPR-Cas, pedigree experiments, preference-performance assays and community ecology to decipher how heliconiine butterflies evolved to be toxic and how this has shaped their interaction with Passiflora plants.

This project resulted in
- Papers:
2 published papers (Biology Letter,s Peer J), 2 submitted (Nat. Com., Ecological Entomology) and 2 in prep (targeting the journals MBE and Molecular Ecology).

- Datasets:
1 transcriptome dataset published in ENA, 1 dataset about the distribution of heliconiine and ithomiine butterflies in the Atlantic Forest published in Zenodo and 1 chemical and fitness dataset published in Dryad. We are also about to published on NCBI and Lepbase a dataset composed of 58 heliconiine genomes, of which 29 were completely new and 10 improved assemblies.

- Contribution to science-training within my institution:
Thesis supervision of 2 bachelor students, 1 master student and several interns in work-experience.

- Fieldwork and Entomological collections:
We visited 9 nature reserves across a 3,000 km latitudinal gradient within the Brazilian Atlantic Forest. We collected samples from 425 butterflies for the host lab collection, which includes records of geographical distribution, wing photos, DNA and chemical extracts. We also collected several butterflies for the entomological collection of UFRN (RN, Brazil) and UNICAMP (SP, Brazil).

- Outreach:
2 outreach activities (Meet the Insects and Minerva Scientifica: Miriam Rothschild).

- Non-scientific communication:
1 publication about the paper de Castro et al. 2021 – Biology Letters.

- Grant:
1 awarded NERC standard grant (Co- Investigator)

A) Biosynthesis and sequestration of toxins in Heliconiines B) Duplication of the CYP405a genes

Distribution of collected heliconiine butterflies withing the Brazilian Atlantic.