Genetic basis, biosynthetic pathways and molecular …...evolution by linking genes, chemicals,...

Kipling Will, UC BerkeleyWendy Moore, U. of ArizonaAman Gill, UC BerkeleyTanya Renner, San Diego State U.Athula Attygalle, Stevens Inst. of Tech.

Funding from the National Science foundationDEB1556957 DEB1556813 DEB1556931 DEB1556898

The Evolution of Geadephagan Chemical Defense

1. Gland cells

I. TISSUE PREPARATION2. Whole body (minus gland cells)

II. HIGH-THROUGHPUT SEQUENCING

Sequence tens of billions of base pairs in 250 bp paired-end reads

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Forward Reverse250 bp 250 bp

1. Threetranscriptomes from gland cells

III. TRANSCRIPTOME ASSEMBLY

contig 1 ≥500 bp

IV. GENE ONTOLOGY ANNOTATION

V. RNASEQ ANALYSES

contig 1

contig 2

GO termsBiological ProcessMolecular FunctionCellular Component

GO termsBiological ProcessMolecular FunctionCellular Component

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Forward Reverse250 bp 250 bp

2. Three transcriptomes from whole body

x3 x3

contig 2 ≥500 bp

Cells 1 Cells 2 Cells 3 Body 1 Body 2 Body 3

Gland-speci c transcript

STEP 2

contig 1contig 2

highlow

tinyurl.com/zfeqj52

Check out the KQED DeepLook video about our NSF research.

Genetic basis, biosynthetic pathways and molecular evolution of carabid quinone production

Carabid beetles are the largest clade of organisms to use a single homologous gland system to produce at least 18 distinct classes of defensive chemical compounds.

Quinone production: independently evolved or ancestrally conserved?

Chemical assays . Beetles are injected with labelled chemical precursors that are incorporated into gland products during synthesis. Using GC-MS the labelled products are analyzed to infer biosynthetic pathways. Steps in the pathways suggest enzymes likely to be involved, clarifying the evolution of biosynthesis at the chemical level and guiding the search for genes underlying quinone production.

What biosynthetic pathways and which genes are used for quinone production?

The chemical composition and delivery mechanisms of carabid defensive compounds are well understood, but little is known about the biosynthetic pathways, genetic architecture, and evolutionary history underlying their production.

Transcriptome sequencing. Gland-only transcriptomes are compared to transcriptomes from whole bodies (minus gland) to identify genes expressed in secretory gland cells during chemical synthesis. Transcripts are functionally annotated and assigned to known metabolic pathways. This information is combined with expression patterns, biosynthesis assays, and data from Tribolium and other systems to identify candidate genes critical for quinone production.

Phylogenetic analysis. four pairs of species, each representing one of four lineages of

among all bombardiers. This will allow us to examine the extent of independent versus conserved evolution, identify possible patterns of convergence and exaptation, and infer the evolutionary history of quinone production, e.g. birth-death dynamics, patterns of selection, and variation in evolutionary rates.

Functional validation of candidate genes using RNAiFunctional assays. Candidate gene expression is knocked down using gene silencing (RNAi) and the resulting gland products are examined, identifying experimentally validated genes with quinone-less or quinone-limited RNAi phenotypes. In a pilot study on Brachinus, knock down of three transcripts reduced expression and resulted in reduced quantities of quinones.

Authors & Funding

some Clivinini

some Chlaenini

Charles Hedgcock, (c) 2015 Wendy Moore

evolution by linking genes, chemicals, biosynthetic pathways and phylogeny in carabid beetles.

We aim to develop an understanding of defensive chemical

RNAi knockdown of three Brachinus genes. based on homology with two genes known to be essential to quinone production in Tribolium castaneum. Knockdown of genes resulted in reduced expression measured via qPCR and absence of defensive toluquinones. ARSB = Arylsulfatase B; MDRP = multi-drug resistance protein, isoform 1 and 2.

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Control RNAi

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Seeking graduate students to study carabid chemical

defense evolution!

Paussinae

Bombardiers

Brachininae

Bombardiers

m-cresol toluhydroquinone toluquinone

tinyurl.com/hlq99ru

Carabidae

Otherbeetles

Quinones

Formic acid

Other chemical classes

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