How RNA interfernce techniques was employed to study the functions of different sequenced genes

2. The Advent of RNA Interference in
Entomology

4. Abstract
‘’RNA interference is a Cellular process by which
mRNA is targeted for degradation by a small
interfering RNA that contains a strand
complementary to a fragment of the target mRNA,
resulting in sequence specific inhibition of gene
expression’’

6.  The discovery of RNAi enabled the use of loss of
function in many non model insects other than
Drosophila to elucidate the roles of specific genes.

7. Introduction
Discovery of RNAi ---------Andrew Z.Fire and Craig C.
Mello in 2006
Currently many RNA-based studies with various insects
are in progress
In this review we will focus on two topics
The current status of RNA experiments in non-
drosophilid insects
The applications of RNAi species-specific insecticides

8. CURRENT STATUS OF RNAI-BASED
EXPERIMENTS IN INSECTS
The advent of RNAi represents a new experimental paradigm beyond
Drosophila
In 1999, Brown et al. (1999) showed that RNAi could be used to phenocopy
the mutations of the Deformed orthologue in the flour beetle, Tribolium
castaneum
Hughes and Kaufman (2000) reported the use of RNAi to dissect gene
function in the development of the milkweed bug, Oncopeltus fasciatus,
which undergoes hemimetabolic maturation.
Since then, many RNAi studies on insects have been reported

10. Milkweed bug, Oncopeltus fasciatus,

11. To review the RNAi-based experiments
reported in non-drosophilid insects
we classify the experiments into four types:
 parental,
embryonic,
larval/nymphal/pupal,
 regeneration-dependent RNAi.

12. Parental RNAi
RNAi can be applied with particular ease in Caenorhabditis elegans,
in which the injection of dsRNA into the body cavity or the application
of dsRNA via ingestion leads to gene inactivation in offspring
embryos
 This RNAi effect found that the injection of dsRNA into the mother’s
hemocoel results in a knockdown of the zygotic genes in the offspring
embryos of T. castaneum ,100% results
In 2004, Liu and Kaufman adopted the technique of paRNAi for use
in milkweed bugs, to determine the role of the gap genes in
segmentation.

13. Shinmyo et al. observed the effects of paRNAi on caudal
development in criket, Gryllus embryos (Fig. 1), where only the
head region is formed in eggs after the injection of dsRNA
targeting Gb’caudal in female crickets.
In the two-spotted spider mite, Tetranychus urticae, paRNAi
against Distalless was used, resulting in phenotypes with
canonical limb truncation as well as the fusion of leg segments

15. Together these findings suggest that paRNAi may function in
many metazoan taxa, indicating that the transmission of dsRNA
across oocyte membranes is a conserved feature of the RNAi
response.
 Since large numbers of RNAi embryos can be readily obtained
with paRNAi and analyzed using standard histochemical and in
situ hybridization procedures, the functional characterization of
genes has been greatly facilitated.

16. Why to use paRNAi?
Thus, the use of paRNAi is an important method for analyzing
early embryogenesis
and it is crucial for many insects whose eggs are not accessible
or do not survive with microinjection
Parental RNAi should allow large-scale RNAi screens in many
species and will greatly facilitate functional approaches in the
burgeoning field of evolutionary developmental biology

17. Embryonic RNAi
When dsRNA for a target gene is injected into developing eggs,
the RNAi phenotypes can appear in embryos, larvae/nymphs, or
even adults. This type of RNAi is called embryonic RNAi
(emRNAi).
 Although using paRNAi is the most efficient way to obtain RNAi
phenotypes in embryos, if a target gene is indispensable for egg
formation, then no eggs can be obtained. In such cases, emRNAi
has been used to analyze gene function during embryogenesis.

18. For instance, Grossmann et al.(2009) studied the role of wingless (wg) in
the leg development of T. castaneum using stage-specific staggered
emRNAi because the paRNAi experiments lead to either empty eggshells
or wild-type cuticles.
In this case, emRNAi was able to circumvent the problem of effects
exhibited during gonad formation or oogenesis. By staggering the
injections, the effects that lead to early embryonic lethality can also be
excluded.
With emRNAi, they demonstrated the separate functions of Tribolium wg
in distal and ventral leg development.

19. Larval/nymphal/pupal RNAi
Tomoyasu and Denell (2004) demonstrated the use of RNAi to
create pupal and adult loss-of-function phenotypes in T.
castaneum by injecting dsRNA into late instar larvae (larval
RNAi, laRNAi).
The laRNAi technique has been used to analyze gene functions
in post-embryonic development to study the molecular basis of
adult morphological diversity in various organisms. For example,
Tomoyasu et al. (2005) used laRNAi in T. castaneum to
determine the function of Ubx/Utx during hindwing/elytron
development.

20. Ubx/Utx RNAi induced a complete transformation of hindwing to
elytron. Unlike Drosophila Ubx, which normally modifies the
development of membranous wings to produce halters, Ubx/Utx in
this beetle seems to promote membranous hindwing development
by repressing elytron identity.
Arakane et al. (2005) demonstrated that laRNAi against laccase 2
in T. castaneum resulted in the formation of white and soft bodies
in a dsRNA dose-dependent fashion.

21. pupal RNAi
Ohnishi et al.(2009) found that pupal RNAi against Bombyx
mori fatty acid transport protein (Bm’FATP) significantly
suppressed the accumulation of cytoplasmic lipid droplets by
preventing the synthesis of triacylglycerols,
which resulted in a significant reduction in the production of the
pheromone bombykol.
In this case, they injected dsRNA into the abdominal tip of 1-
day-old pupae.
After injection, the pupae were maintained under normal
conditions until adult emergence.

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

23. Martin et al. (2006) found that the injection of dsRNA into the
hemocoel of nymphs and adults of the cockroach, Blattella
germanica, can be used to silence gene function in vivo. They
used nyRNAi to elucidate the function of RXR/USP, which is one
component, along with EcR, of the heterodimeric nuclear receptor
of 20-hydroxyecdysone (20E).
They demonstrated that Bg’RXR knockdown nymphs progressed
through the instar correctly, but development was arrested at the
end of this stage, making them unable to molt into adults. These
results suggested that RXR/USP function, in relation to molting, is
conserved across the insect Class.

24. Nymphal RNAi against Gb’par-1, which leads to mortality in G. bimaculatus due to
defects in ecdysis. Such a gene may be a target for RNAi to induce mortality. (a, b)
A cricket nymph of negative control (a) and mortal cricket nymphs of Gb’par-1
nyRNAi nymphs (b). Bar represents 1 mm.

25. The phenotype of nymphal RNAi (nyRNAi) against Gryllus bimaculatus laccase 2
lacks tanning (top). This result indicates that the expression of laccase 2 is crucial for
cuticle tanning in G. bimaculatus. After injection of the dsRNA for laccase 2 into
nymphs at the fifth instar, the nymphs were reared to be adults. The bottom shows a
control cricket. Bar represents 1 cm.

26. Hamada et al. (2009) performed loss-of-function analyses on G. bimaculatus genes
homologous to the human genes that are responsible for certain human disorders:
fragile X mental retardation 1 (fmr1) and Dopamine receptor (DopR).
For Gb’fmr1, they observed three major nyRNAi phenotypes:
(i) abnormal wing postures;
(ii) abnormal calling song; and
(iii) loss of the circadian locomotor rhythm;
indicating that the cricket has the potential to become a novel model system to explore
human neuronal pathogenic mechanisms and to screen therapeutic drugs with RNAi.
The conservation of systemic RNAi in these insects suggests that this pathway can be
exploited for the gene-specific manipulation of larvae/nymphs and pupae/adults in a
wide range of insects.

27. Regeneration-dependent RNAi
Nymphs of hemimetabolous insects, such as cockroaches and
crickets, exhibit a remarkable capacity for regenerating complex
structures from damaged legs (for review see Nakamura et al.
2008).
Currently, our group is the only one working on insect leg
regeneration with RNAi. Using the cricket, G. bimaculatus, as a
model, Nakamura et al. (2008) found that RNAi phenotypes can
be observed during regeneration following the amputation of legs.

28. Because no phenotype is induced by nyRNAi in an intact cricket leg, this effect is
designated as regeneration-dependent RNAi (rdRNAi).
Since that time, the functions of various genes encoding signaling factors and
cellular adhesion proteins, such as Fat and Dachsous, have been investigated
during Gryllus leg regeneration.
Mito et al. (2002) showed that Gryllus orthologues of Drosophila hedgehog (Gb’hh),
wingless (Gb’wg) and decapentaplegic (Gb’dpp) are expressed during leg
regeneration and play essential roles in the establishment of the proximal-distal axis
 To confirm the roles of Gb’wg during regeneration, Nakamura et al.(2007) used
rdRNAi and found that no regeneration took place when Gb’armadillo (the
orthologue of b-catenin) was knocked down. However, no phenotypes were
observed when Gb’wg was knocked down

29. 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

31. Applications of RNAi For Development of
Species-specific dsRNA Insecticides
A serious problem for insecticides is that they can kill non-targeted animals.
To address this issue, the possibility of using RNAi to kill only the target
animals by down-regulating essential gene functions in insects has been
recognized for many years (Price & Gatehouse 2008)
Bando et al. screened Gryllus target genes to develop G. bimaculatus-
specific dsRNA insecticides (Fig. 4). However, this method was considered
unfeasible because the method relies on the injection of dsRNA into insects,
which is not possible for practical application of insecticides. A more effective
method may be to use a bait containing dsRNA, as developed in nematodes
through feeding which can uptake dsRNA through feeding.

32. Because dsRNA may be degraded in the gut, it seemed that the knockdown
of a target gene was unlikely.
 However, Turner et al. (2006) demonstrated that in the horticultural pest,
Epiphyas postvittana (Lepidoptera: Tortricidae), RNAi could be triggered by
the oral delivery of dsRNA to larvae.
MeyeringVos and Müller (2007) found that treatment of the adult cricket, G.
bimaculatus, by injection or ingestion of a dsRNA for sulfakinin, a group of
brain/gut neuropeptides, led to a stimulation of food intake, indicating that the
uptake of dsRNA in the Gryllus occurs in the gut. Although these results
suggest that the oral delivery of dsRNA is feasible, the problem of the
continuous feeding needed to use dsRNA, as an insecticide remained
unsolved.

33. To circumvent this problem, Baum et al. (2007) made transgenic corn
plants engineered to express dsRNAs for the western corn root-worm
(WCR).
 The plants showed a significant reduction in WCR feeding damage in a
growth chamber assay, suggesting that the RNAi pathway can be
exploited to control insect pests via the in planta expression of a dsRNA.
 Mao et al. (2007) also made Arabidopsis thaliana and Nicotiana tobacum
transgenic plants expressing dsRNA specific to a cytochrome P450 gene
(CYP6AE14) of the cotton bollworm (Helicoverpa armigera), which
permits this herbivore to tolerate the cotton metabolite, gossypol.

34. When larvae are fed the transgenic plant, larval growth is retarded due to
inhibitory effects of gossypol. Thus, they concluded that feeding insects with
plant material expressing dsRNA may be a general strategy for the delivery of
RNAi and could find applications in entomological research and field control
of insect pests .
Whyard et al. harnessed the sequence specificity of RNAi to design orally-
delivered dsRNAs that selectively killed target insects.
 They found that D. melanogaster, T. castaneum, pea aphids (Acyrthosiphon
pisum), and tobacco hornworms (Manduca sexta) were selectively killed
when fed species-specific dsRNA targeting vacuolar-type ATPase transcripts.

35. Efficiency of RNAi depends on Developmental
Stage sand Species
Bellés (2010) listed the insect species studied with RNAi in vivo in his
comprehensive review and pointed out that the efficiency of systemic
RNAi in vivo depends on the species; less-derived species are
generally more sensitive than more-derived species.
 Tomoyasu et al. (2008) performed a genome wide survey to compare
genes involved in the machinery of RNAi between Tribolium and C.
elegans, both of which show a robust systemic RNAi response.
They found significant differences between the genes involved in the
RNAi machinery of these organisms.

36. Thus, they concluded that insects might use an alternative mechanism for
the systemic RNAi response.
Because over-expression of dsRNAs within cells using hairpin RNAs
readily triggers RNAi in D. melanogaster tissues resistant to systemic RNAi
Bellés (2010) speculated that the poor sensitivity is not related to the RNAi
core machinery, but rather to the penetration and transmission of the
interfering signal through cells and tissues and to the occurrence of
degradation mechanisms that are able to remove alien

37. Huvenne and Smagghe (2010) reviewed the mechanisms of
dsRNA uptake. They list two major types of mechanisms:
(i) the transmembrane channel-mediated uptake mechanism,
in which orthologs of C. elegans systemic RNAi defective
mutant gene (sid) may be involved; and
(ii) the endocytosis-mediated uptake mechanism, in which
vacuolar H+-ATPase, clathrin, scavenger receptors, etc. may
be involved.

38. If this is the case, one of the ways to circumvent the problem of
low RNAi efficiency is to use chemically modified siRNAs or
carriers which may stabilize siRNAs and transport them into
cells, as investigated for human RNAi therapy (Tiemann & Rossi
2009).
Using such methods in insects could help to render other insects
amenable to systemic RNAi and may influence pest control
approaches

39. Future Prospects For RNAI-Based Experiments
in Insects
RNAi-based experiments have provided several interest-ing results and shed light on
gene functions in non-drosophilid insects.
The potential application of RNAi techniques to any gene and any species could lead to
comparative studies for the function of a gene, or gene network, in species covering a
large spectrum of insect orders (Bellés 2010). Moreover, RNAi can facilitate
comparative studies and evolutionary insight into other processes, such as social
behaviors, reproductive strategies, and host-parasite interactions
Systemic RNAi-based genome-wide screening is particularly useful to identify genes
involved at the whole animal level, e.g. in deter-mination of life span and size, metabolic
controls, cir-cadian clock systems, ecdyses, etc.