Effect of using<i>green fluorescent protein</i>double-stranded RNA as non-target negative control in<i>Nasonia vitripennis</i>RNA interference assays

Rougeot, Julien; Wang, Yidong; Verhulst, Eveline C.; Nevels, Michael

doi:10.1017/exp.2020.67

Cited by 6 publications

(4 citation statements)

References 21 publications

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“…In this study, we used GFP as a non-target exogenous control based on the absence of this gene in Myxozoa genomic data, although this gene is endogenously present in free-living Cnidaria 61 as well as other GFP-like homologs across Metazoa 62 . While several reports regarding off-target or indirect effects of GFP on gene expression in RNAi studies exist 63 – 65 , we anticipated that using GFP as non-target control for RNAi-based studies in our model species S . molnari can be employed.…”

Section: Discussionmentioning

confidence: 99%

RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari

Kyslík,

Born-Torrijos,

Holzer

et al. 2024

Sci Rep

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RNA interference (RNAi) is an effective approach to suppress gene expression and monitor gene regulation. Despite its wide application, its use is limited in certain taxonomic groups, including cnidarians. Myxozoans are a unique group of cnidarian parasites that diverged from their free-living ancestors about 600 million years ago, with several species causing acute disease in farmed and wild fish populations. In this pioneering study we successfully applied RNAi in blood stages of the myxozoan Sphaerospora molnari, combining a dsRNA soaking approach, real-time PCR, confocal microscopy, and Western blotting. For proof of concept, we knocked down two unusual actins, one of which is known to play a critical role in S. molnari cell motility. We observed intracellular uptake of dsRNA after 30 min and accumulation in all cells of the typical myxozoan cell-in-cell structure. We successfully knocked down actin in S. molnari in vitro, with transient inhibition for 48 h. We observed the disruption of the cytoskeletal network within the primary cell and loss of the characteristic rotational cell motility. This RNAi workflow could significantly advance functional research within the Myxozoa, offering new prospects for investigating therapeutic targets and facilitating drug discovery against economically important fish parasites.

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Section: Discussionmentioning

confidence: 99%

RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari

Kyslík,

Born-Torrijos,

Holzer

et al. 2024

Sci Rep

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“…Curiously, GFP dsRNAi appears to have a generally upregulating effect as opposed to WT controls despite for n -alkene quantities in males, which has not been reported before. However, a recent study hints at potential off-target effects of GFP dsRNAi on a small subset of N. vitripennis genes mainly involved in microtubule and sperm development ( Rougeot et al, 2021 ). Interestingly, this appears to be also the case for the expressions of fas1 and fas2 gene transcripts, which are significantly upregulated in the GFP dsRNAi controls, again exclusively in males ( Figure 2—figure supplement 1B ).…”

Section: Discussionmentioning

confidence: 99%

“…The Quick-RNA Tissue/Insect Kit (Zymo Research, Freiburg, Germany) was used to purify the resulting dsRNA product. GFP (green fluorescent protein) dsRNA, which presumably has no known targets in the Nasonia genome ( Rougeot et al, 2021 ), was used as a control. GFP dsRNA was synthesized from the vector pOPINEneo-3C-GFP, which was kindly donated by Ray Owens (Addgene plasmid #53534; http://n2t.net/addgene :53534; RRID: Addgene_53534 ).…”

Section: Methodsmentioning

confidence: 99%

Decoding the genetic and chemical basis of sexual attractiveness in parasitic wasps

Sun

Lange

Gadau

et al. 2023

eLife

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Attracting and securing potential mating partners is of fundamental importance for reproduction. Therefore, signaling sexual attractiveness is expected to be tightly coordinated in communication systems synchronizing senders and receivers. Chemical signaling has permeated through all taxa of life as the earliest and most widespread form of communication and is particularly prevalent in insects. However, it has been notoriously difficult to decipher how exactly information related to sexual signaling is encoded in complex chemical profiles. Similarly, our knowledge of the genetic basis of sexual signaling is very limited and usually restricted to a few case studies with comparably simple pheromonal communication mechanisms. The present study jointly addresses these two knowledge gaps by characterizing two fatty acid synthase genes, that most likely evolved by tandem gene duplication, simultaneously impacting sexual attractiveness and complex chemical surface profiles in parasitic wasps. Gene knock-down in female wasps dramatically reduces their sexual attractiveness coinciding with a drastic decrease in male courtship and copulation behavior. Concordantly, we found a striking shift of methyl-branching patterns in the female surface pheromonal compounds, which we subsequently demonstrate to be the main cause for the greatly reduced male response. Intriguingly, this suggests a potential coding mechanism for sexual attractiveness mediated by specific methyl-branching patterns in CHC profiles. So far, the genetic underpinnings of methyl-branched CHCs are not well understood despite their high potential for encoding information. Our study sheds light on how biologically relevant information can be encoded in complex chemical profiles and on the genetic basis of sexual attractiveness.

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“…The Quick-RNA Tissue/Insect Kit (Zymo Research, Freiburg, Germany) was used to purify the resulting dsRNA product. GFP (Green Fluorescent Protein) dsRNA, which has no known targets in the Nasonia genome (50), was used as a control. GFP dsRNA was synthesized from the vector pOPINEneo-3C-GFP, which was kindly donated by Ray Owens (Addgene plasmid #53534; http://n2t.net/addgene:53534; RRID:Addgene_53534).…”

Section: Methodsmentioning

confidence: 99%

Decoding the genetic and chemical basis of sexual attractiveness in parasitic wasps

Sun

Lange

Gadau

et al. 2023

Preprint

View full text Add to dashboard Cite

Attracting and securing potential mating partners is of fundamental importance for successfully initiating reproduction and thus assuring the passing of genes to the next generation. Therefore, signaling sexual attractiveness is expected to be tightly coordinated in communication systems synchronizing senders and receivers. Chemical signaling has permeated through all taxa of life as the earliest and most wide-spread form of communication and is particularly prevalent in insects. However, it has been notoriously difficult to decipher how exactly information related to sexual signaling is encoded in complex chemical profiles. Similarly, our knowledge of the genetic basis of sexual signaling is very limited and usually restricted to a few case studies with comparably simple pheromonal communication mechanisms. The present study jointly addresses these two knowledge gaps by characterizing a single gene simultaneously impacting sexual attractiveness and complex chemical surface profiles in parasitic wasps. Knocking down a fatty acid synthase gene in female wasps dramatically reduces their sexual attractiveness coinciding with a drastic decrease in male courtship and copulation behavior. Concordantly, we found a striking shift of methyl-branching patterns in the female surface pheromonal compounds, which we subsequently demonstrate to be the main cause for the greatly reduced male response. Intriguingly, this suggests a potential coding mechanism for sexual attractiveness mediated by specific methyl-branching patterns, whose genetic underpinnings are not well understood despite their high potential for encoding information. Our study sheds light on how biologically relevant information can be encoded in complex chemical profiles and on the genetic basis of sexual attractiveness.

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Effect of usinggreen fluorescent proteindouble-stranded RNA as non-target negative control inNasonia vitripennisRNA interference assays

Cited by 6 publications

References 21 publications

RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari

RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari

Decoding the genetic and chemical basis of sexual attractiveness in parasitic wasps

Decoding the genetic and chemical basis of sexual attractiveness in parasitic wasps

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