The advent of RNA interference in Entomology

Justus Liebig University, Giessen (Germany)

By: Ravi Dhiman Harmeet Singh chawla

Introduction: Outline

What is RNAi?

Brief History

Pathways of RNAi

RNAi -Beginning in Insects

What Is RNAi?

RNA interference (RNAi) is a highly evolutionarily conserved mechanism of gene regulation

There are two main classes of small non-coding RNAs (sRNAs) found in insects namely microRNAs (miRNAs) and short interfering RNAs (siRNAs)

The biogenesis of miRNAs and siRNAs differs in the way they enter the RNAi pathway, both have common elements

miRNAs and siRNAs are generated from larger more complex dsRNA by the ribonuclease III enzyme Dicer

Both then associates with an Argonaut family protein (AGO) to become part of the RISC- which functions to down regulate gene expression

Need for RNAi

Defense Mechanism– As a defense mechanism to protect against transposons and

other insertional elements

– Protects against viral infections

Genome Wide Regulation– RNAi plays a role in regulating development and genome

maintenance

Brief History of RNAi

Source: Invitrogen

siRNA PathwaymiRNA Pathway

Plants Animals Plants and Animals

RNAi -Beginning in Insects

Andrew Fire and Craig Mello first described the RNAi phenomenon in the nematode worm C.elegans. Injected into C.elegans dsRNA led to an efficient sequence-specific silencing.

And coined the term "RNA Interference"

RNAi can operate in

insects too!

RNAi was found to work in many diverse species

Fungi

Trypanosomes

Zebrafish

Mice

Fire and Mello Experiment

Source: nobleprize.org

Andrew Fire Craig Mello

Happy Ending

RNAi-mediated gene knockdown

RNAi has been readily utilised to investigate gene function in insects

Effective RNAi has been demonstrated in many insect orders Diptera (Lum et al., 2003; Dietzl et al., 2007) Lepidoptera (Chen et al., 2008; Yu et al., 2008; Tian et al.,

2009; Terenius et al., 2011) Coleoptera (Arakane et al., 2004; Suzuki et al., 2008) Hymenoptera (Schluns & Crozier, 2007; Antonio et al.,

2008) But has also been performed in insects in a variety of

developmental stages

RNAi in Insects

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Parental RNAi

From Shinmyo Y, Mito T, Matsushita T et al. (2005) caudal is required for gnathal and thoracic patterning and for posterior elongation in the intermediate-germband cricket Gryllus bimaculatus. Mechanisms of Development 122, 231–239

Shinmyo et al. (unpublished data) observed the effects of paRNAi on caudal development in Gryllus embryos where only the head region is formed in eggs after the injection of dsRNA targeting Gb’caudal in female crickets.

Bucher et al.(2002) found that parental RNAi was highly efficient in Tribolium

Nearly 100% of embryos in the first egg laid after injection had RNAi phenotypes.

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Large numbers of RNAi embryos can be readily obtained with paRNAi

Crucial for many insects whose eggs are not accessible or do not survive with microinjection (Bucher et al.2002).

Investigations in the field of evolutionary developmental biology

Why Parental RNAi?

From Gregor Bucher, Johannes Scholten, Martin Klingle. (2002) Parental RNAi in Tribolium (Coleoptera) Current biology ,Volume 12, Issue 3, R85–R86 14

Embryonic RNAi

RNAi construct targeting the gene of interest is injected into developing eggs

From Grossmann D, Scholten J, Prpic NM (2009) Separable functions of wingless in distal and ventral patterning of the Tribolium leg . Development Genes and Evolution 219, 469–479

Grossmann et al. (2009) found that paRNAi experiments lead to either empty egg shells or wild-type cuticles.emRNAi was able to circumvent the problem of effects exhibited during gonad formation or oogenesis. The effects that lead to early embryonic lethality was also excluded.

Why Embryonic RNAi?

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Larval/nymphal /pupal RNAi

Analyze gene functions in postembryonic development to study the molecular basis of adult morphological diversity in various organisms.

Useful for analyzing the functions of enzymes in biosynthesis pathways.

Nymphal RNAi (nyRNAi) was reported by Dong and Friedrich (2005) as a systemic RNAi mediated gene knockdown in the juvenile grasshopper, Schistocerca americana.

From G. bimaculatus. Bando et al. (unpublished results)

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Regeneration-dependent RNAi

Nymphs of hemimetabolous insects, such as cockroaches and crickets, exhibit a remarkable capacity for regenerating complex structures from damaged legs

No phenotype is induced by nyRNAi in an intact cricket leg

Bando et al. (2009) used a cricket leg model to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration.

From Bando T, Mito T, Maeda Y et al. (2009) Regulation of leg size and shape by the Dachsous/Fat signalling pathway duringregeneration. Development 136, 2235–2245

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APPLICATIONS OF RNAi FOR DEVELOPMENT OF SPECIES-SPECIFIC dsRNA INSECTICIDES

Pros:Provides an environmental friendly approach to generate plants resistant to viruses and insects

kill only the target animals by down-regulating essential gene functions

Cons:Relies on the injection of dsRNA into insects, which is not possible for practical application of insecticides.

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EFFICIENCY OF RNAI DEPENDS ON DEVELOPMENTAL STAGES AND SPECIES

The dependence of RNAi efficiency on developmental stages or species is attributed to

Differences in the expression patterns of the sid orthologs (C.elegans systemic RNAi defective mutant gene )

Differences in the sensitivity of dsRNA detection systems for endocytosis in insect cells

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FUTURE PROSPECTS FOR RNAI-BASED EXPERIMENTS IN INSECTS

The mechanisms underlying RNAi phenomena, such as species or tissue dependent changes in the sensitivity to dsRNAs, are not fully understood.

Systemic RNAi-based genome-wide screening is particularly useful to identify genes involved at the whole animal level,

The insect world, with its more than 30 orders, about 1 million described species, and perhaps 30 million still awaiting taxonomic discovery, still hides many genetic black boxes, and RNAi appears to be a formidable key for opening a large number of them.

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Thank You……..Any questions?

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