Lacewing venom: Linking the molecular and phenotypic evolution of adaptive traits

Project Information

VenomEvolvability

Grant agreement ID: 101039862

DOI

10.3030/101039862

EC signature date 3 February 2022

Start date 1 March 2022

End date 28 February 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 971,00

EU contribution

€ 1 499 971,00

Coordinated by

UNIVERSITETET I OSLO
Norway

Periodic Reporting for period 1 - VenomEvolvability (Lacewing venom: Linking the molecular and phenotypic evolution of adaptive traits)

Reporting period: 2022-03-01 to 2024-08-31

Summary of the context and overall objectives of the project

Understanding the ability of species to adapt to their environment, or their ‘evolvability’, is central to evolutionary biology. Most traits are complex in that their phenotype results from the contributions of many genes with small, sometimes non-additive effects. While quantitative genetics has been instrumental in showing that short term evolvability depends on additive genetic variation, it ignores details of the molecular underpinnings of phenotypic characters that are crucial for the production and maintenance of additive genetic variation, and therefore evolvability at longer time scale. This impacts our understanding of both evolvability itself and how it relates to macro-evolutionary processes, and calls for model traits that enable the integration of quantitative and molecular genetics.

Animal venoms are great model systems for understanding the link between molecular and phenotypic evolution. Venom phenotypes such as toxicity result from the combined action of a relatively small number of secreted proteins, or ‘toxins’, whose effect on fitness can be characterised. In addition to their molecular components (i.e. toxins), venoms are accompanied by venom producing tissues, delivering structures, and specialised behaviours associated with venom use. Together, these traits form integrated venom systems whose phenotypes can be measured within clearly defined adaptive contexts such as predation or defence. Venoms thus provide an excellent opportunity to study the evolution of traits across a wide range of biological scales, from their origin as phenotypic novelties to their evolution as adaptations.

This project focuses on the venom of Neuroptera, an insect order that includes antlions and lacewings and comprises approximately 6500 known species across 16 families. While adults are non-venomous, most have carnivorous larvae that capture and ingest prey using paralysing and liquefying venom whose composition remains virtually unknown. Some species also have short generation times (25 days), and are easily bred in the lab. Neuroptera thus provides an opportunity to study the origins of phenotypic novelties and the evolution of their genotypic–phenotypic relationships from molecular to morphological levels across evolutionary timescales.

The project seeks to identify the molecular underpinnings of venom evolvability and venom trait evolution across micro- and macroevolutionary timescales by following three complementary aims:

AIM 1: MOLECULAR GENETIC BASIS OF VENOM EVOLVABILITY
Test whether venom traits exhibit greater evolvability than non-venom traits, and whether these differences in evolvability is due to modularity of traits across different levels of biological complexity.

AIM 2: ECOLOGICAL FACTORS DRIVING THE ADAPTIVE EVOLUTION OF VENOM
Test whether venom evolution is primarily driven by selection due to extrinsic ecological factors, toxin functional diversity is generated through phenotypically neutral evolutionary processes, or venom adaptation is mainly due to phenotypic plasticity.

AIM 3: MACROEVOLUTION OF THE NEUROPTERAN VENOM SYSTEM
Examine the molecular evolutionary processes and mechanisms involved in the emergence and evolution of neuropteran venoms as phenotypic novelties, and test whether the molecular underpinnings of evolvability of venom traits also facilitate the rate of evolution of venom and whether the molecular underpinnings of venom phenotypic novelties also facilitate venom evolvability.

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

The project thus far has focused mainly on Aims 1 and 3.

For Aim 1, we have completed sample collection for transcriptional evolvability and sequenced tissues for regulatory network analyses. To facilitate accurate measurements of gene expression we have, in collaboration with the Earth Biogenome Project Norway (EBP-Nor), generated and annotated a chromosome-level reference genome for the heterogametic sex (male) from our focal population of C. carnea.

For Aim 3, we have collected samples from species that represent a broad sampling across Neuroptera as well as the distribution of C. carnea. For venom system comparisons between neuropteran families, we have generated 3D reconstructions of venom delivery structures, musculature, and venom glands using µCT, including a redescription of the venom system. We have in collaboration with EBP-Nor completed three reference-quality genomes from Neuroptera (Chrysopa perla, Myrmeleon formicarius, in addition to the afore-mentioned Chrysoperla carnea), while Osmylus fulvicephalus is being sequenced in collaboration with the Biodiversity Genomics Europe (BGE) consortium.

To provide a solid foundation for the remaining experiments, our first scientific deliverable (manuscript in final stages of preparation) provides the first comprehensive insight into the venom of Neuroptera via C. carnea. Using high-quality genomes from a male and female, long-read-based reference transcriptomes from their offspring across each life stage, and venom proteomics, we provide a complete paralog, allele, and splice-variant resolved venom proteome. These data revealed surprisingly few examples of large toxin gene family expansions, that alternative splicing provides a mechanism that facilitates exon-specific neofunctionalization of co-opted genes, and that the only previously characterised toxin described from Neuroptera (a bacterially derived chaperonin) is not a significant venom component. We also found that the most abundant toxin is an ABC-toxin-like protein secreted via a non-canonical secretory pathway, that was obtained by horizontal gene transfer (HGT) from a commensal bacterium. Our results contrast the canonical mechanisms of venom evolution and highlight the importance of studying neglected taxa for understanding mechanisms involved in the evolution of molecular novelties.

As part of our genome annotation efforts, we have made a bioinformatic pipeline that uses a combination of proteomic and functional annotation to identify toxin sequences from transcriptome data, and where applicable map these onto a reference genome to identify paralogs, allelic variants, and splice-forms. The pipeline will be made available via GitHub upon publication of the manuscript on the detailed description of C. carnea venom.

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)

The most significant achievements listed above go beyond the state-of-the art by i) completely redefining the neuropteran venom system, ii) providing a complete allele and splice-variant resolved venom proteome that lets us determine the exact mechanisms underlying the biochemical diversity found in the venom, and iii) showing the involvement of rare mechanisms of emergence of novelty and evolutionary innovation in the evolution of venom (splicing and HGT) including multiple occasions, timing, and identification of a putative donor of a toxin that originated from horizontal gene transfer and is secreted via a non-canonical secretory pathway.

Apart from the above, the main achievements and progress thus far provide a solid foundation for analyses performed in the near future that will advance venom research and evolutionary biology beyond state-of-the-art. These include detailed insights in to regulatory mechanisms governing potential specialised production of protein spliceforms on a cellular level, regulatory mechanisms underlying venom plasticity, and the most comprehensive set of estimates to date of evolvability across levels of biological complexity comprising a complex phenotype.