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| Status |
Public on Dec 30, 2020 |
| Title |
Single cell sequencing reveals that constitutive activation of cellular immunity underlies the evolution of resistance to infection |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Goal: Natural populations of Drosophila melanogaster vary widely in their capacity to resist infection by the parasitic wasp Leptopilina boulardi. To date, little is known about the genetic, cellular and molecular basis underpinning the variation in susceptibility to parasitic wasps. In D.melanogaster populations artificially selected for parasitoid resistance, an increase in the number of circulating hemocytes (blood cells) is observed. One possibility is that this is accompanied by changes in hemocyte activation state, resulting in a successful defence response when exposed to parasitic wasps. We tested this possibility by generating populations that are highly resistant and highly susceptible to the parasitic wasps. We generated these populations after 26 generations of experimental evolution, starting from a single interbred wild caught population. Here, we study how selection for resistance to the parasitic wasp L. boulardi changes the number and type of circulating hemocytes in D. melanogaster larvae using scRNA-seq.
Methods: Wild caught D. melanogaster females were collected from Cambridge, UK, to establish an outbreed population. From this, three replicated populations were selected for resistance to L. boulardi strain NSRef for 26 generations (Selection). Another three populations were maintained in the laboratory for 26 generations without being exposed to parasitoid-associated selection pressures (No Selection). At generation 26, second instar D. melanogaster larvae (48-63 hours after fertilization) from each population were infected with L. boulardi for three hours (Infection) or maintained without infection (No infection). 48 hours after, circulating hemocytes from third instar D. melanogaster larvae (96-111 hours after fertilization) from each population were collected in PBS and cleaned in OptiPrep solution (1.09g/ml). 10X Single Cell GEX v2 libraries were prepared and sequenced. CellRanger v2 was used to generate sample cell count matrices. Seurat v3 was used to integrate, normalise and cluster the cell types.
Results: We identified novel plasmatocyte cell types, immature and mature lamellocytes and crystal cells. We identify a single lineage of cellular differentiation starting from a self-cycling plasmatocyte population, enriched for genes involved in extracellular matrix organisation, and ending in a mature lamellocyte population that has enriched expression of genes involved in signalling, hemostasis and gluconeogenesis. Immature lamellocytes were overrepresented in populations selected for resistance to parasitoids. Infection with parasitoid resulted in an increase in both immature and mature lamellocytes in circulation.
Conclusions: Selection for resistance to the parasitic wasps resulted in the constitutive activation of the D. melanogaster immune system, where plasmatocytes differentiate into immature lamellocytes even where they are not exposed to parasitic wasps. We speculate these constitutive changes assist in mounting a successful defence response when exposed to parasitic wasps.
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| Overall design |
Two selection regimes (Selection and No selection) and two infection treatments (Infection and No infection), three samples for each condition (12 total). All 12 samples were multiplexed on each lane of NovaSeq 6000 S1. Two lanes were sequenced in total, i.e. each sample was sequenced over two lanes at 1/12 depth in each lane.
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| Contributor(s) |
Leitão A, Ramesh A, Day J, Jiggins F |
| Citation(s) |
33357377 |
| Submission date |
Apr 17, 2020 |
| Last update date |
Jan 01, 2021 |
| Contact name |
Frank Jiggins |
| E-mail(s) |
fmj1001@cam.ac.uk
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| Phone |
+441223333175
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| Organization name |
Department of Genetics, University of Cambridge
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| Street address |
Downing Street
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| City |
Cambridge |
| ZIP/Postal code |
CB2 3EH |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL25244 |
Illumina NovaSeq 6000 (Drosophila melanogaster) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA625925 |
| SRA |
SRP256887 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE148826_RAW.tar |
295.5 Mb |
(http)(custom) |
TAR (of MTX, TSV) |
| GSE148826_intergrated_hemocytes.Robj.gz |
630.1 Mb |
(ftp)(http) |
ROBJ |
| GSE148826_intergrated_lamellocyte_subcluster.Robj.gz |
483.8 Mb |
(ftp)(http) |
ROBJ |
| GSE148826_intergrated_rna_count_matrix.csv.gz |
28.8 Mb |
(ftp)(http) |
CSV |
| GSE148826_intergrated_scaled_hvg_matrix.csv.gz |
311.6 Mb |
(ftp)(http) |
CSV |
| GSE148826_intergrated_subcluster_scaled_hvg_matrix.csv.gz |
242.8 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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