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DrosDel Immunity Panel

The DrosDel Immunity Panel (B. Lemaitre Lab)

contact: bruno.lemaitre@epfl.ch

 

The ‘DrosDel Immunity’ Panel (DD-Im) is a set of Drosophila lines useful for studying the immune system in a controlled genetic background, thereby minimizing non-specific effects.

The lines were generated by the Luis Teixeira and Bruno Lemaitre labs (notably, also Mark Hanson for effector mutants). Mutations in these flies were introgressed by successive backcrosses in the DrosDel background using the scheme described in Ferreira et al.(1), or sometimes directly generated in the DrosDel background by CRISPR/Cas9.

This panel includes flies:

  • lacking functional Toll (spzrm7) or Imd (RelE20) pathways.
  • with no melanization: PPO1, PPO2
  • deficient for phagocytosis and hemocyte sessility: NimC1, eater
  • lacking various immune effectors: Bomanins, Transferrin 1, antimicrobial peptides….
  • lacking 14 antimicrobial peptide (AMP) genes or lacking non-overlapping subsets of these 14 AMPs (group A, B, C) as well as group D flies (Daisho and Baramicin A). These can be conveniently used to identify an AMP involved in a process (See ref (2)).

 

While these isogenized lines can be useful to study the immune response in a rather controlled background, we recommend using alternative methods to confirm the results (rescue, RNAi, analyzing the mutations over a deficiency, or analyzing the same or another mutation in another background). We cannot exclude that significant portions of the genome are not well isogenized (notably in close proximity to the mutation of interest), or that resulting phenotypes come from complex interactions between the mutation and second site mutation of the DrosDel background -- not to mention varying status of white and white+ rescue, GFP, DsRed, etc... We hope to expand this collection of immunity fly strains.

 

 

Stock and isogenization details

 

VDRC_ID

Code

Genotype

Original reference

Isogenization reference

Chr

Remarks

349900

BL1

w[1118]; ; RelE20

(3, 4)

(1)

III

A deletion of Relish (which also affects a nearby gene). The “ebony” marker of the original stock (Dan Hultmark‘s lab) was removed  by recombination with an Oregon stock. The RelE20 mutation was then introgressed in the DrosDel background. RelE20 lack a functional Imd pathway and are susceptible to Gram negative bacterial infection. The transcription factor Relish may also be involved in the immune defense against viruses downstream of cGas-like, Sting and IKK (5).

349901

BL2

w[1118]; ; spzrm7/TM6C, Sb1

(6, 7)

(1)

III

spzrm7 (also known as spz4) is a genetically null mutation in spz (generated by EMS during Nusslein-Volhard and Wieschaus screen). Several markers of the original stock (M317 Tubingen stock center) including ebony were removed by recombination (7). spzrm7 mutants are homozygous viable, female sterile. This mutation blocks the activation of the Toll pathway. Note that the Toll pathway might be activated independently of spz by spz5(8).

349902

BL3

w[1118]; PPO1Δ, PPO2Δ

(9, 10)

unpublished

II

PPO1Δ and PPO2Δ mutations were generated by homologous recombination (PPO1 gene is replaced by a w+ cassette). PPO1Δ, PPO2Δ show no hemolymphatic melanization after injury. PPO3 in lamellocytes can still contribute to melanization around wasp eggs (10).

349903

BL4

w[1118]; NimC1Δ; eaterΔ

(11, 12)

unpublished

II, III

NimC1Δ and eaterΔ mutations were generated by homologous recombination (both genes are replaced by a w+ cassette). NimC1Δ, eaterΔ have strongly reduced phagocytosis ability and defective hemocyte sessility due to the absence of eater. Whilst NimC1Δ, eaterΔ larvae have more hemocytes, NimC1Δ, eaterΔ adults tend to have decreased hemocyte number over time. This line can be used to assess the function of phagocytosis, although it likely impacts other processes. Of note, the non-iso NimC1, Eater flies are not isogenized in the DrosDel background due to developing sterility problems.

349904

BL5

w[1118], Hayan-psh[Def]

(13)

unpublished

I

Hayan-psh[Def] is a short deletion generated by Shu Kondo removing two serine proteases and strongly reduces both the melanization and the Toll pathways.

349905

BL6

w[1118], Hayan-psh[Def]; NimC1Δ; eaterΔ, RelE20/TM6C, Sb1

 

unpublished

I, II, III

w[1118], Hayan-psh[Def]; NimC1Δ; eaterΔ, RelE20 (also called ΔITPM) are viable but are extremely immune deficient, having both reduced Toll pathway and Melanization (due to Hayan-psh[Def]), no phagocytosis (due to NimC1Δ; eaterΔ) and no Imd pathway (RelE20). This stock is maintained with a balancer on the third chromosome (TM6C). NimC1Δ and eaterΔ mutations were generated by homologous recombination (both genes are replaced by a w+ cassette).

349906

BL7

w[1118] iso; iso; iso

(14)

This line was cleared of Nora virus

 

The DrosDel wild-type (isogenized for chr 1;2;3, with w[1118] line) used as reference. A contaminating Nora virus was discovered in this stock that may have affected survival data in studies prior to 2021. Nora virus infection is associated with slightly increased mortality upon infection (anecdotally ~15%), and greatly reduced lifespan (see (15)). The DrosDel w[1118] isogenic genotype may be particularly susceptible to Nora virus compared to other backgrounds (e.g. OregonR). However, Nora virus-free survival and lifespan are similar to or better than OregonR.

349907

BL8

w[1118]; ; Sp7[SK6]

(10, 17, 18, 19)

(10)

III

A null mutation affecting the Sp7/MP2 serine protease that regulates melanization in Drosophila, isogenized in the w[1118] DrosDel background. Sp7 strongly blocks the blackening reaction at the injury site of larvae but has less effect in adults for blackening (10).

349908

BL9

w[1118], Hayan[SK6]

(20)

(10)

I

A null mutation affecting the Hayan serine protease that regulates melanization in Drosophila, isogenized in the w[1118], DrosDel background.

349909

BL10

w[1118]; ; PGRP-SD[SK1]

(21)

(21)

III

A null mutation affecting the secreted pattern recognition receptor PGRP-SD that promotes the activation of the Imd pathway upstream of PGRP-LC, isogenized in the w[1118], DrosDel background.

349910

BL11

w[1118]; ; PGRP-LB[Δ]

(22)

(21)

III

A null mutation affecting PGRP-LB, which encodes a secreted negative regulator of the Imd pathway upstream of PGRP-LC, isogenized in the w[1118], DrosDel background.

349911

BL12

ΔAMP10: (w[1118]; DefSK3, AttC[Mi], Dro-AttAB[SK2], Mtk[R1], Dpt[SK1]/CyO; Drs[R1], AttD[SK1]/ TM6C, Sb1

(2)

(2)

II, III

A fly line with 7 deletions removing 10 Antimicrobial peptides genes, all isogenized in the w[1118], DrosDel background. DroAtt removes Drosocin locus (two peptides (23)) and Attacin-A and -B. The MtkR1 and DrsR1 loci each carry one w+ transgene, and the AttCMi mutation also causes GFP to be expressed in the eyes, ocelli, and occasionally the gut.

349912

BL13

ΔAMP14: w[1118]; DefSK3, AttC[Mi], Dro-AttAB[SK2], Mtk[R1], Dpt[SK1]/CyO; Drs[R1], AttD[SK1], ΔCecA-C/TM6C, Sb1

(2, 24)

(2, 24)

II, III

A fly line with 8 deletions removing 14 Antimicrobial peptides genes (ΔAMP10 + deletion of Cecropin locus), all isogenized in the w[1118], DrosDel background. DroAtt removes Drosocin locus (two peptides (23)) and Attacin-A and -B.  The MtkR1 and DrsR1 loci each carry one w+ transgene, and the AttCMi mutation also causes GFP to be expressed in the eyes, ocelli, and occasionally the gut. The CecA-C mutation also produces DsRed in the eyes, ocelli, and abdomen.

349913

BL14

ΔAMP Group A: w[1118]; DefSK3;  ΔCecA-C/TM6C, Sb1

(2)

(2)

II, III

A fly line with 2 deletions removing the Defensin and the Cecropin Locus (4 genes), all isogenized in the w[1118], DrosDel background. The polymorphism at the Buletin locus is Alanine.

349914

BL15

ΔAMP Group C: w[1118]; Mtk[R1]; Drs[R1]

(2)

(2)

II, III

A fly line with 2 mutations removing 2 antifungal peptides (2 genes), all isogenized in the w[1118], DrosDel background.

349915

BL16

ΔAMP Group B: w[1118]; AttC[Mi], DroAtt[SK2], Dpt[SK1]; AttD[SK1]

(2)

(2)

II

A fly line with 4 mutations removing the 2 Diptericin, the 4 Attacins and Drosocin, all isogenized in the w[1118], DrosDel background. DroAtt removes the Drosocin locus (2 peptides, (23)) and Attacin-A and -B (Hanson et al., 2022; Proc R Soc B).

349916

BL17

w[1118]; ; ΔCecA-C

(2)

(2)

III

A deletion removing the Cecropin Locus (4 genes), all isogenized in the w[1118], DrosDel background.

349917

BL18

w[1118]; ; AttD[SK1]

(2)

(2)

III

A mutation affecting Attacin-D, isogenized in the w[1118], DrosDel background.

349918

BL19

w[1118]; Dro[SK4]

(2)

(2)

II

A mutation affecting the whole Dro locus, isogenized in the w[1118], DrosDel background.

349919

BL20

w[1118], Tsf1[JP94]

(25)

(26)

I

A mutation affecting the Tsf1 gene, isogenized in the w[1118], DrosDel background.

349920

BL21

w[1118]; ΔBaraA{DsRed}

(27)

(27)

II

A fly line deleted for the whole BaramicinA locus, isogenized in the w[1118] Drosdel background. This mutation retains the full gene region of CG30059, but does replace sequence directly downstream with a DsRed construct. Note that this genotype is lacking both CG18279 (BaraA1) and CG33470 (BaraA2), and also lacks CG18278 as the mutation was generated in a background that never had the BaraA1 + CG18278 locus duplication in the first place. i.e. generated in a background with only CG33470 and CG30059.

349921

BL22

w[1118]; DptA[Δ822] (derived from DGRP-822)

(28)

(28)

II

A premature stop mutation affecting the DptA locus, isogenized in the w[1118], DrosDel background. This mutation was derived from the strain DGRP-822 and backcrossed into the DrosDel background.

349922

BL23

w[1118]; DptB[KO]

(29)

(28)

II

A deletion removing the DptB locus (29), isogenized in the w[1118], DrosDel background (FlyBase link). This line induces the related gene DptA to a lesser extent compared to the DrosDel background, and so within this immune mutant panel, it may most accurately be considered a hypomorph for DptA alongside being fully null for DptB (28).

349923

BL24

w[1118]; DptA[S69R-4]

(28)

(28)

II

A fly line with DptA carrying the S69R polymorphism (derived from DGRP-38), isogenized in the w[1118], DrosDel background.

349924

BL25

w[1118]; ; Drs[R1]

(2)

(2)

III

A deletion removing the Drs locus (generated by homologous recombination, containing a w+ gene), isogenized in the w[1118], DrosDel background.

349925

BL26

w[1118]; Mtk[R1]

(2)

(2)

II

A deletion removing the Mtk locus (generated by homologous recombination, containing a w+ gene), isogenized in the w[1118], DrosDel background.

349926

BL27

w[1118]; Dpt[SK1]/CyO

(2)

(2)

II

A deletion removing DptA and DptB, isogenized in the w[1118], DrosDel background. This mutation is homozygous viable, and so CyO may be absent.

349927

BL28

w[1118]; Dro-AttAB[SK2]

(2)

(2)

II

A deletion removing the Drc locus and AttA and AttB, isogenized in the w[1118], DrosDel background.

349928

BL29

w[1118]; Bom55CΔ/CyO

(30)

(30)

II

A deletion removing 10 genes at the Bomanin 55C locus, isogenized in the w[1118], DrosDel background. The deletion was generated by Clemmons et al. (29). (CyO floating).

349929

BL30

w[1118]; AttC[Mi]

(2)

(2)

II

A Minos insertion affecting Attacin-C, isogenized in the w[1118], DrosDel background.

349930

BL31

w[1118]; ; LysB-PΔ

(31)

(31)

III

A fly line with an 11.5-kb deletion, removing LysC (a putative pseudogene) and the 4 lysozyme genes (i.e., Lys B, LysD, LysE, and Lys P) that are known to be strongly induced in the gut. Isogenized in the w[1118], DrosDel background.

349931

BL32

w[1118]; Def[SK3]

(2)

(2)

II

A deletion removing the Defensin gene, first published in Parvy et al. (2019; eLife) having been backcrossed into the w[1118] DrosDel background. A revisiting of this allele in Hanson et al. (2022; Proc R Soc B) later realized the Def[SK3]-containing chromosome had an alternate version of the Buletin allele, and so this was further isogenized to ensure the Buletin locus had an alanine allele matching the w[1118], DrosDel isogenic background, and the resulting strain published in Hanson et al. (2023; Dis Mod Mech). The correction of the Buletin allele to be alanine (matching the DrosDel background) affects the interpretation of Def[SK3] effects in previous literature pertaining to antibacterial defense in minor ways.

349932

BL33

w[1118]; ΔDaisho

(32)

(15)

II

A deletion removing the 2 Daisho genes (32). This deletion was later isogenized into the w[1118], DrosDel isogenic background (15).

349933

BL34

Group D: w[1118]; ΔBaraA, ΔDaisho

 

unpublished

II

A fly line with 2 mutations removing the Baramicin A gene(s) and the 2 Daisho genes. This strain carries 2 copies of a DsRed construct (from ΔBaraA and ΔDaisho).

349934

BL35

w[1118]; ; TotAZ[SK6]

(33)

(33)

III

A fly line, called TotAZ, with a deletion removing the TotA, TotB, TotC and TotZ genes, isogenized in the w[1118], DrosDel background.

349935

BL36

w[1118]; TotM[jp1621]; TotA-Z[SK6], TotX[jp44]

(33)

(33)

II, III

A fly line, called TotXMAZ, carrying 1 mutation affecting TotM and 1 mutation affecting TotX and a deletion removing the TotA, TotB, TotC and TotZ genes, isogenized in the w[1118], DrosDel background.

References

  1. Ferreira, et al., The Toll-dorsal pathway is required for resistance to viral oral infection in Drosophila. PLoS pathogens 10, e1004507 (2014). DOI: 10.1371/journal.ppat.1004507
  2. A. Hanson et al., (2019) Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach. eLife 8:e44341 (2019). DOI: 10.7554/eLife.44341
  3. Hedengren, et al., Relish, a central factor in the control of humoral but not cellular immunity in Drosophila. Mol Cell 4, 827–37 (1999). DOI: 10.1016/s1097-2765(00)80392-5
  4. Leulier, A. Rodriguez, R. S. Khush, J. M. Abrams, B. Lemaitre, The Drosophila caspase Dredd is required to resist gram-negative bacterial infection. EMBO Rep 1, 353–8 (2000). DOI: 10.1093/embo-reports/kvd073
  5. Cai, C. Meignin, J.-L. Imler, cGAS-like receptor-mediated immunity: the insect perspective. Curr Opin Immunol 74, 183–189 (2022). DOI: 10.1016/j.coi.2022.01.005
  6. Morisato, K. Anderson, The spätzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo. Cell 76, 677–688 (1994). DOI: 10.1016/0092-8674(94)90507-x
  7. Lemaitre, E. Nicolas, L. Michaut, J.-M. Reichhart, J. A. Hoffmann, The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973–983 (1996). DOI: 10.1016/s0092-8674(00)80172-5
  8. Nonaka, et al., Characterization of Spz5 as a novel ligand for Drosophila Toll-1 receptor. Biochemical and Biophysical Research Communications 506, 510–515 (2018). DOI: 10.1016/j.bbrc.2018.10.096
  9. Binggeli, C. Neyen, M. Poidevin, B. Lemaitre, Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila. PLoS Pathogens 10, e1004067 (2014). DOI: 10.1371/journal.ppat.1004067
  10. P. Dudzic, S. Kondo, R. Ueda, C. M. Bergman, B. Lemaitre, Drosophila innate immunity: regional and functional specialization of prophenoloxidases. BMC Biology 13 (2015). DOI: 10.1186/s12915-015-0193-6
  11. J. Bretscher, et al., The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster. Biology Open 4, 355–63 (2015). DOI: 10.1242/bio.201410595
  12. Melcarne, et al., Two Nimrod receptors, NimC1 and Eater, synergistically contribute to bacterial phagocytosis in Drosophila melanogaster. The FEBS Journal 286(14):2670-2691 (2019). DOI: 10.1111/febs.14857
  13. P. Dudzic, M. A. Hanson, I. Iatsenko, S. Kondo, B. Lemaitre, More Than Black or White: Melanization and Toll Share Regulatory Serine Proteases in Drosophila. Cell Rep 27, 1050-1061.e3 (2019). DOI: 10.1016/j.celrep.2019.03.101
  14. Ryder, et al., The DrosDel deletion collection: a Drosophila genomewide chromosomal deficiency resource. Genetics 177, 615–29 (2007). DOI: 10.1534/genetics.107.076216
  15. A. Hanson, B. Lemaitre, Antimicrobial peptides do not directly contribute to aging in Drosophila, but improve lifespan by preventing dysbiosis. Disease Models & Mechanisms 16, dmm049965 (2023). DOI: 10.1242/dmm.049965
  16. Li, S. Rommelaere, S. Kondo, B. Lemaitre, Renal Purge of Hemolymphatic Lipids Prevents the Accumulation of ROS-Induced Inflammatory Oxidized Lipids and Protects Drosophila from Tissue Damage. Immunity 52, 374-387.e6 (2020). DOI: 10.1016/j.immuni.2020.01.008
  17. S. Ayres, D. S. Schneider, A Signaling Protease Required for Melanization in Drosophila Affects Resistance and Tolerance of Infections. PLoS Biol 6, e305 (2008). DOI: 10.1371/journal.pbio.0060305
  18. Castillejo-López, U. Häcker, The serine protease Sp7 is expressed in blood cells and regulates the melanization reaction in Drosophila. Biochemical and Biophysical Research Communications 338, 1075–1082 (2005). DOI: 10.1016/j.bbrc.2005.10.042
  19. Tang, Z. Kambris, B. Lemaitre, C. Hashimoto, Two proteases defining a melanization cascade in the immune system of drosophila. J Biol Chem 281, 28097–104 (2006). DOI: 10.1074/jbc.M601642200
  20. J. Nam, I. H. Jang, H. You, K. A. Lee, W. J. Lee, Genetic evidence of a redox-dependent systemic wound response via Hayan protease-phenoloxidase system in Drosophila. The EMBO Journal 31, 1253–65 (2012). DOI: 10.1038/emboj.2011.476
  21. Iatsenko, S. Kondo, D. Mengin-Lecreulx, B. Lemaitre, PGRP-SD, an Extracellular Pattern-Recognition Receptor, Enhances Peptidoglycan-Mediated Activation of the Drosophila Imd Pathway. Immunity 45, 1013–1023 (2016). DOI: 10.1016/j.immuni.2016.10.029
  22. C. Paredes, D. P. Welchman, M. Poidevin, B. Lemaitre, Negative Regulation by Amidase PGRPs Shapes the Drosophila Antibacterial Response and Protects the Fly from Innocuous Infection. Immunity 35, 770–779 (2011). DOI: 10.1016/j.immuni.2011.09.018
  23. A. Hanson, S. Kondo, B. Lemaitre, Drosophila immunity: the Drosocin gene encodes two host defence peptides with pathogen-specific roles. Proc Biol Sci 289, 20220773 (2022). DOI: 10.1098/rspb.2022.0773
  24. Carboni, M. A. Hanson, S. A. Lindsay, S. A. Wasserman, B. Lemaitre, Cecropins contribute to Drosophila host defense against fungal and Gram-negative bacterial infection. Genetics 220(1):iyab188 (2022). DOI: 10.1093/genetics/iyab188
  25. Iatsenko, A. Marra, J.-P. Boquete, J. Peña, B. Lemaitre, Iron sequestration by transferrin 1 mediates nutritional immunity in Drosophila melanogaster. Proc Natl Acad Sci USA 117, 7317–7325 (2020). DOI: 10.1073/pnas.1914830117
  26. Marra, F. Masson, B. Lemaitre, The iron transporter Transferrin 1 mediates homeostasis of the endosymbiotic relationship between Drosophila melanogaster and Spiroplasma poulsonii. microLife 2, uqab008 (2021). DOI: 10.1093/femsml/uqab008
  27. A. Hanson, et al., The Drosophila Baramicin polypeptide gene protects against fungal infection. PLoS Pathog 17, e1009846 (2021). DOI: 10.1371/journal.ppat.1009846
  28. A. Hanson, L. Grollmus, B. Lemaitre, Ecology-relevant bacteria drive the evolution of host antimicrobial peptides in Drosophila. Science 381, eadg5725 (2023). DOI: 10.1126/science.adg5725
  29. Barajas-Azpeleta, et al., Antimicrobial peptides modulate long-term memory. PLOS Genetics 14, e1007440 (2018). DOI: 10.1371/journal.pgen.1007440
  30. W. Clemmons, S. A. Lindsay, S. A. Wasserman, An Effector Peptide Family Required for Drosophila Toll-Mediated Immunity. PLOS Pathogens 11, e1004876 (2015). DOI: 10.1371/journal.ppat.1004876
  31. Marra, M. A. Hanson, S. Kondo, B. Erkosar, B. Lemaitre, Drosophila Antimicrobial Peptides and Lysozymes Regulate Gut Microbiota Composition and Abundance. mBio e0082421 (2021). DOI: 10.1128/mBio.00824-21
  32. B. Cohen, S. A. Lindsay, Y. Xu, S. J. H. Lin, S. A. Wasserman, The Daisho Peptides Mediate Drosophila Defense Against a Subset of Filamentous Fungi. Front Immunol 11, 9 (2020). DOI: 10.3389/fimmu.2020.00009
  33. Rommelaere, et al., A humoral stress response protects Drosophila tissues from antimicrobial peptides. Current Biology 34(7):1426-1437.e6. (2024). DOI: 10.1016/j.cub.2024.02.049

What are the Heidelberg CFD CRISPR toolbox (HD_CFDtools) lines?

The Heidelberg CFD CRISPR Library contains a set of toolbox stocks (HD_CFDtools), including a series of UAS-Cas9 lines containing an upstream open reading frame (uORF) of varying length from XS to XXL. These produce a range of expression levels of Cas9 and enable high gene editing activity without detectable toxicity or artefacts that can potentially be caused by high levels of Cas9. The longer the uORF, the lower the Cas9 expression, so the most appropriate Cas9 line to use for an experiment may be carefully selected based on the desired expression level.

To facilitate your experiments further, some lines have already been created to combine UAS-Cas9 with common Gal4 driver lines (e.g. act-, hh-, nub-, ptc-, GMR-, dpp- and vg-Gal4). Such stocks can be crossed to transgenic sgRNA lines to induce conditional CRISPR mutagenesis in Gal4 expressing cells.

Lines for induction of Cas9 expression by FLP-out are also available. Cas9 can be induced in all Gal4 expressing cells or only in a random subset, with the latter approach resulting in fluorescently marked mosaics. Such mosaics can be a powerful method to analyze neighbouring mutant and wildtype cells in the same tissue.

How do I acknowledge use of these lines?

When using lines from the DrosDel Immunity Panel (DD-Im), please cite the relevant original and/or isogenization reference and use the VDRC_ID identifier for each stock in your publications.

Additionally, please acknowledge the VDRC for distributing fly lines. A simple statement is sufficient and can either be placed in the Materials and Methods section or in the Acknowledgements.

Suggested format:
Transgenic fly stocks and/or plasmids were obtained from the Vienna Drosophila Resource Center (VDRC, www.vdrc.at).