RNA interference in the cat flea, Ctenocephalides felis: Approaches for sustained gene knockdown and evidence of involvement of Dicer-2 and Argonaute2

Catriona H Edwards, John Baird, Erich Zinser, Debra J Woods, Sophie Shaw, Ewan M Campbell, Alan S Bowman (Corresponding Author)

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Abstract

Effective RNA interference (RNAi) methods have been developed in many pest species enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80% of glutathione S-transferase σ (GSTσ) was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65% knockdown of GSTσ or 60% Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administering of dsRNA in whole blood via a membrane feeding system resulting in 96% knockdown of GSTσ within 2 days and sustained up to, at least, 7 days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3 hr after exposure to exogenous dsRNA implicating the short-interfering RNA pathway. This study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight how the gene knockdown response progresses.
Original languageEnglish
Pages (from-to)993-1002
Number of pages10
JournalInternational Journal for Parasitology
Volume48
Issue number13
Early online date24 Sep 2018
DOIs
Publication statusPublished - Nov 2018

Fingerprint

Ctenocephalides
Gene Knockdown Techniques
RNA Interference
Double-Stranded RNA
Glutathione Transferase
Pest Control
Microinjections
Small Interfering RNA
Up-Regulation
Membranes
Mortality

Keywords

  • RNA interference
  • Ctenocephalides felis
  • GST
  • Dicer-2
  • Argonaute2
  • nucleases
  • artificial feeding
  • knockdown

Cite this

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title = "RNA interference in the cat flea, Ctenocephalides felis: Approaches for sustained gene knockdown and evidence of involvement of Dicer-2 and Argonaute2",
abstract = "Effective RNA interference (RNAi) methods have been developed in many pest species enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80{\%} of glutathione S-transferase σ (GSTσ) was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65{\%} knockdown of GSTσ or 60{\%} Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administering of dsRNA in whole blood via a membrane feeding system resulting in 96{\%} knockdown of GSTσ within 2 days and sustained up to, at least, 7 days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3 hr after exposure to exogenous dsRNA implicating the short-interfering RNA pathway. This study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight how the gene knockdown response progresses.",
keywords = "RNA interference, Ctenocephalides felis, GST, Dicer-2, Argonaute2, nucleases, artificial feeding, knockdown",
author = "Edwards, {Catriona H} and John Baird and Erich Zinser and Woods, {Debra J} and Sophie Shaw and Campbell, {Ewan M} and Bowman, {Alan S}",
note = "This work was supported by a Knowledge Transfer Network BBSRC Industrial Case (# BB/L502467/1) studentship in association with Zoetis Inc. We acknowledge the expert help provided by Nikki Kernell (Zoetis Inc., Kalamazoo) during the feeding trials. Declarations of interest: none.",
year = "2018",
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doi = "10.1016/j.ijpara.2018.04.006",
language = "English",
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pages = "993--1002",
journal = "International Journal for Parasitology",
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T1 - RNA interference in the cat flea, Ctenocephalides felis

T2 - Approaches for sustained gene knockdown and evidence of involvement of Dicer-2 and Argonaute2

AU - Edwards, Catriona H

AU - Baird, John

AU - Zinser, Erich

AU - Woods, Debra J

AU - Shaw, Sophie

AU - Campbell, Ewan M

AU - Bowman, Alan S

N1 - This work was supported by a Knowledge Transfer Network BBSRC Industrial Case (# BB/L502467/1) studentship in association with Zoetis Inc. We acknowledge the expert help provided by Nikki Kernell (Zoetis Inc., Kalamazoo) during the feeding trials. Declarations of interest: none.

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N2 - Effective RNA interference (RNAi) methods have been developed in many pest species enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80% of glutathione S-transferase σ (GSTσ) was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65% knockdown of GSTσ or 60% Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administering of dsRNA in whole blood via a membrane feeding system resulting in 96% knockdown of GSTσ within 2 days and sustained up to, at least, 7 days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3 hr after exposure to exogenous dsRNA implicating the short-interfering RNA pathway. This study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight how the gene knockdown response progresses.

AB - Effective RNA interference (RNAi) methods have been developed in many pest species enabling exploration of gene function. Until now RNAi had not been attempted in the cat flea, Ctenocephalides felis, although the development of RNAi approaches would open up potential avenues for control of this important pest. This study aimed to establish if an RNAi response occurs in adult C. felis upon exposure to double-stranded RNA (dsRNA), which administration methods for dsRNA delivery could bring about effective gene knockdown and to investigate dynamics of any RNAi response. Knockdown of 80% of glutathione S-transferase σ (GSTσ) was achieved by intrahaemoceolic microinjection of dsGSTσ but this invasive technique was associated with relatively high mortality rates. Immersing C. felis in dsGSTσ or dsDicer-2 overnight resulted in 65% knockdown of GSTσ or 60% Dicer-2, respectively, and the degree of knockdown was not improved by increasing the dsRNA concentration in the bathing solution. Unexpectedly, the greatest degree of knockdown was achieved with the continuous administering of dsRNA in whole blood via a membrane feeding system resulting in 96% knockdown of GSTσ within 2 days and sustained up to, at least, 7 days. Thus, unlike in many other species, the gut nucleases do not impair the RNAi response to ingested dsRNA in C. felis. A modest, but significant, upregulation of Dicer-2 and Argonaute2 was detectable 3 hr after exposure to exogenous dsRNA implicating the short-interfering RNA pathway. This study represents the first demonstration of experimentally induced RNAi in the cat flea as well as giving insight how the gene knockdown response progresses.

KW - RNA interference

KW - Ctenocephalides felis

KW - GST

KW - Dicer-2

KW - Argonaute2

KW - nucleases

KW - artificial feeding

KW - knockdown

U2 - 10.1016/j.ijpara.2018.04.006

DO - 10.1016/j.ijpara.2018.04.006

M3 - Article

VL - 48

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EP - 1002

JO - International Journal for Parasitology

JF - International Journal for Parasitology

SN - 0020-7519

IS - 13

ER -