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The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

Schmitt-Engel C, Schultheis D, Schwirz J, Ströhlein N, Troelenberg N, Majumdar U, Dao VA, Grossmann D, Richter T, Tech M, Dönitz J, Gerischer L, Theis M, Schild I, Trauner J, Koniszewski ND, Küster E, Kittelmann S, Hu Y, Lehmann S, Siemanowski J, Ulrich J, Panfilio KA, Schröder R, Morgenstern B, Stanke M, Buchhholz F, Frasch M, Roth S, Wimmer EA, Schoppmeier M, Klingler M, Bucher G - Nat Commun (2015)

Bottom Line: Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions.RNAi screens in other organisms promise to reduce this bias.This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: 1] Johann-Friedrich-Blumenbach-Institut, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany [2] Department Biologie, Entwicklungsbiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstraße 5, 91058 Erlangen, Germany.

ABSTRACT
Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

No MeSH data available.


Related in: MedlinePlus

Postembryonic phenotypes.(a) Wild-type ovary stained for F-actin (red) and DNA (blue). Pro-oocytes (asterisks) become encapsulated by somatic follicle cells and separated by stalk cells (arrow). (d) Upon Tc-MED24 RNAi, egg chambers are misarranged, not separated by stalk cells (arrow) and subsequently they fuse (IV). (b,c) After Tc-retained-RNAi the three most distal antennomeres (1–3) of the adult antenna are fused. (e,f) RNAi against Tc-ATP7 led to strongly reduced odoriferous gland content and partially melanized secretions (white arrowheads; remnant of posterior abdominal cuticle marked by open arrowhead:). (g–j) The knockdown of iB_04887 led to wing blisters in Tribolium pupae (arrowhead in h). Transgenic RNAi against the Drosophila ortholog showed the same phenotype, revealing a novel candidate for integrin mediated adhesion (arrowhead in j). Scale bars indicate 100 μm in (a–f) and 1 mm in (g–j).
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f5: Postembryonic phenotypes.(a) Wild-type ovary stained for F-actin (red) and DNA (blue). Pro-oocytes (asterisks) become encapsulated by somatic follicle cells and separated by stalk cells (arrow). (d) Upon Tc-MED24 RNAi, egg chambers are misarranged, not separated by stalk cells (arrow) and subsequently they fuse (IV). (b,c) After Tc-retained-RNAi the three most distal antennomeres (1–3) of the adult antenna are fused. (e,f) RNAi against Tc-ATP7 led to strongly reduced odoriferous gland content and partially melanized secretions (white arrowheads; remnant of posterior abdominal cuticle marked by open arrowhead:). (g–j) The knockdown of iB_04887 led to wing blisters in Tribolium pupae (arrowhead in h). Transgenic RNAi against the Drosophila ortholog showed the same phenotype, revealing a novel candidate for integrin mediated adhesion (arrowhead in j). Scale bars indicate 100 μm in (a–f) and 1 mm in (g–j).

Mentions: Odoriferous stink glands play crucial roles in insect defence and communication but are not present in Drosophila5960. In the iBeetle screen, we identified 32 genes with relevant phenotypes, including the absence of the gland contents, altered colour and composition of secretions, or melanosis. Interestingly, only 5 among these 32 genes showed an enrichment of greater than fourfold in odoriferous gland transcriptomes compared with mid-abdominal tissues60, illustrating that a phenotypic screen can only partially be replaced by transcriptomic approaches (see Supplementary Fig. 4). For instance, the Tc-copper-transporting-ATPase-I (Tc-ATP7) is neither upregulated nor downregulated (Supplementary Fig. 5) but RNAi mediated knockdown (iB_02517) caused a reduced gland content and melanosis phenotype (Fig. 5f) and a loss of benzoquinones (Supplementary Fig. 6). Another emerging field is the shaping of the adult body during typical holometabolous insect metamorphosis were larval epidermal cells are reused24. For instance, Tc-retained led to rounded female genitalia and the fusion of distal antennal segments (Fig. 5c). Interestingly, these anatomical features vary in Tenebrionids61, making this gene a good candidate for morphological evolutionary studies. Finally, one difference between Tribolium telotrophic oogenesis and Drosophila meroistic oogenesis is that in Tribolium germ line stem cells stop proliferating at larval stages while the somatic stem cell lineage remains active throughout life23. In Drosophila, both lineages remain active and dependent on each other, making it difficult to study the somatic lineage independently. In the iBeetle screen we identified several genes probably required for the somatic lineage, such as Tc-MED24 whose knockdown led to incomplete separation of egg chambers and a reduced number of follicle cells (Fig. 5d).


The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

Schmitt-Engel C, Schultheis D, Schwirz J, Ströhlein N, Troelenberg N, Majumdar U, Dao VA, Grossmann D, Richter T, Tech M, Dönitz J, Gerischer L, Theis M, Schild I, Trauner J, Koniszewski ND, Küster E, Kittelmann S, Hu Y, Lehmann S, Siemanowski J, Ulrich J, Panfilio KA, Schröder R, Morgenstern B, Stanke M, Buchhholz F, Frasch M, Roth S, Wimmer EA, Schoppmeier M, Klingler M, Bucher G - Nat Commun (2015)

Postembryonic phenotypes.(a) Wild-type ovary stained for F-actin (red) and DNA (blue). Pro-oocytes (asterisks) become encapsulated by somatic follicle cells and separated by stalk cells (arrow). (d) Upon Tc-MED24 RNAi, egg chambers are misarranged, not separated by stalk cells (arrow) and subsequently they fuse (IV). (b,c) After Tc-retained-RNAi the three most distal antennomeres (1–3) of the adult antenna are fused. (e,f) RNAi against Tc-ATP7 led to strongly reduced odoriferous gland content and partially melanized secretions (white arrowheads; remnant of posterior abdominal cuticle marked by open arrowhead:). (g–j) The knockdown of iB_04887 led to wing blisters in Tribolium pupae (arrowhead in h). Transgenic RNAi against the Drosophila ortholog showed the same phenotype, revealing a novel candidate for integrin mediated adhesion (arrowhead in j). Scale bars indicate 100 μm in (a–f) and 1 mm in (g–j).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525174&req=5

f5: Postembryonic phenotypes.(a) Wild-type ovary stained for F-actin (red) and DNA (blue). Pro-oocytes (asterisks) become encapsulated by somatic follicle cells and separated by stalk cells (arrow). (d) Upon Tc-MED24 RNAi, egg chambers are misarranged, not separated by stalk cells (arrow) and subsequently they fuse (IV). (b,c) After Tc-retained-RNAi the three most distal antennomeres (1–3) of the adult antenna are fused. (e,f) RNAi against Tc-ATP7 led to strongly reduced odoriferous gland content and partially melanized secretions (white arrowheads; remnant of posterior abdominal cuticle marked by open arrowhead:). (g–j) The knockdown of iB_04887 led to wing blisters in Tribolium pupae (arrowhead in h). Transgenic RNAi against the Drosophila ortholog showed the same phenotype, revealing a novel candidate for integrin mediated adhesion (arrowhead in j). Scale bars indicate 100 μm in (a–f) and 1 mm in (g–j).
Mentions: Odoriferous stink glands play crucial roles in insect defence and communication but are not present in Drosophila5960. In the iBeetle screen, we identified 32 genes with relevant phenotypes, including the absence of the gland contents, altered colour and composition of secretions, or melanosis. Interestingly, only 5 among these 32 genes showed an enrichment of greater than fourfold in odoriferous gland transcriptomes compared with mid-abdominal tissues60, illustrating that a phenotypic screen can only partially be replaced by transcriptomic approaches (see Supplementary Fig. 4). For instance, the Tc-copper-transporting-ATPase-I (Tc-ATP7) is neither upregulated nor downregulated (Supplementary Fig. 5) but RNAi mediated knockdown (iB_02517) caused a reduced gland content and melanosis phenotype (Fig. 5f) and a loss of benzoquinones (Supplementary Fig. 6). Another emerging field is the shaping of the adult body during typical holometabolous insect metamorphosis were larval epidermal cells are reused24. For instance, Tc-retained led to rounded female genitalia and the fusion of distal antennal segments (Fig. 5c). Interestingly, these anatomical features vary in Tenebrionids61, making this gene a good candidate for morphological evolutionary studies. Finally, one difference between Tribolium telotrophic oogenesis and Drosophila meroistic oogenesis is that in Tribolium germ line stem cells stop proliferating at larval stages while the somatic stem cell lineage remains active throughout life23. In Drosophila, both lineages remain active and dependent on each other, making it difficult to study the somatic lineage independently. In the iBeetle screen we identified several genes probably required for the somatic lineage, such as Tc-MED24 whose knockdown led to incomplete separation of egg chambers and a reduced number of follicle cells (Fig. 5d).

Bottom Line: Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions.RNAi screens in other organisms promise to reduce this bias.This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: 1] Johann-Friedrich-Blumenbach-Institut, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany [2] Department Biologie, Entwicklungsbiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstraße 5, 91058 Erlangen, Germany.

ABSTRACT
Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

No MeSH data available.


Related in: MedlinePlus