They found that the injection of dsRNA corresponding to the vermilion eye color gene of first instar nymphs triggered a suppression of ommochrome formation in the eye lasting through two instars, equivalent to 10–14 days.
This study provided an important insight about how regenerating blastemal cells are aware of both their position and the normal size of the leg. Because the Ds/Ft system is conserved in vertebrates, their results provided clues to the mechanisms of regeneration, which are relevant to vertebrate systems (Bando et al. 2009).
1. The advent of RNA interference
Justus Liebig University, Giessen
By: Ravi Dhiman
Harmeet Singh chawla
2. Introduction: Outline
What is RNAi?
Pathways of RNAi
RNAi -Beginning in Insects
3. 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
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
4. Need for RNAi
– 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
5. Brief History of RNAi
6. siRNA Pathway
Plants Animals Plants and Animals
7. 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
And coined the term "RNA Interference"
8. RNAi was found to work in many diverse
9. Fire and Mello Experiment
10. Andrew FireCraig Mello
11. RNAi-mediated gene knockdown
RNAi has been readily utilised to investigate gene function in
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
12. RNAi in Insects
13. 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.
14. 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
Why Parental RNAi?
From Gregor Bucher, Johannes Scholten, Martin Klingle. (2002) Parental RNAi in Tribolium (Coleoptera) Current biology ,Volume 12, Issue 3,
15. 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?
16. 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)
17. 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
From Bando T, Mito T, Maeda Y et al. (2009) Regulation of leg size
and shape by the Dachsous/Fat signalling pathway during
regeneration. Development 136, 2235–2245
18. APPLICATIONS OF RNAi FOR DEVELOPMENT
OF SPECIES-SPECIFIC dsRNA INSECTICIDES
Provides an environmental
friendly approach to generate plants
resistant to viruses and insects
kill only the target animals by
down-regulating essential gene
Relies on the injection of dsRNA
into insects, which is not possible for
practical application of insecticides.
19. 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
Differences in the sensitivity of dsRNA detection
systems for endocytosis in insect cells
20. 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.