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Proc. Arthropod. Embryol. Soc. Jpn. 47, 57–58 (2012)Ⓒ 2012 Arthropodan Embryological Society of Japan
ISSN 1341–1527

Difference in Efficiency of RNA Interference during Embryonic
Development in the Sawfly, Athalia rosae ruficornis: Embryonic RNAi
versus Parental RNAi*Naotoshi YOSHIYAMA1, 2) and Masatsugu HATAKEYAMA2)
Department of Geosphere and Biosphere Science, Graduate School of Science and Technology, Shinshu University, Matsumoto,
Nagano 390–8621, Japan
2)
Division of Insect Sciences, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305–8634, Japan
E-mail: sawfly@affrc.go.jp (MH)1)

Gene knockdown by introducing double-stranded
RNA (dsRNA), known as RNA interference (RNAi), hasbeen widely used for gene functional analysis in a varietyof insects. The RNAi in insects is categorized as embryonicRNAi, larval RNAi and parental RNAi based on the timingof the introduction of dsRNA (Tomoyasu et al., 2008;
Huvenne and Smagghe, 2010; Terenius et al., 2011).
Embryonic RNAi, in which dsRNA is injected into eggs or
early embryos, targets genes expressed during
embryogenesis, and larval RNAi, in which dsRNA is
injected into larvae or nymphs, targets genes expressed inlarval and/or larval-adult transition stages. Parental RNAi
induces gene knockdown in the next generation byinjecting dsRNA into mothers. These RNAi methods areconvenient and applicable to non-model species withlimited genetic and genomic background; however, theefficiency of RNAi effects are largely dependent on thespecies, targeting genes and stages of dsRNA introduction(Terenius et al., 2011). Embryonic RNAi is effective in awide variety of insects and has been shown to be the mostversatile method. On the other hand, larval RNAi andparental RNAi although are effective in several species,
they do not work well in such model species as the fruit fly,Drosophila melanogaster and the silkworm, Bombyx mori(Miller et al., 2008; Tomoyasu et al., 2008; Terenius et al.,2011). Coleoptera and Hymenoptera are susceptible toRNAi at various developmental stages (Beye et al., 2002;
Bucher et al., 2002; Tomoyasu and Denell, 2004; Niimi etal., 2005; Kuwayama et al., 2006; Lynch and Desplan, 2006;
Miller et al., 2008; Werren et al., 2009). We have reported
that the sawfly, Athalia rosae ruficornis, belonging to thelower suborder of Hymenoptera shows RNAi responses inmost developmental stages (Sumitani et al., 2005;Yoshiyama et al., 2010). Embryonic RNAi and parental
RNAi are both applicable to functional analysis of genes
expressed during embryonic development, although it is
not known if there is difference in the efficiency ofknockdown effects between these two methods. Here we
examined the effectiveness of embryonic RNAi andparental RNAi by targeting an integrated constitutively
active green fluorescent protein (GFP) gene and an
endogenous Distal-less (Dll) gene.
We first compared the effects of embryonic RNAi andparental RNAi targeting the GFP transgene. Forembryonic RNAi a 298-bp-long dsRNA targeting the GFP
transgene was injected into mature eggs taken fromfemales homozygous for the GFP transgene. The GFP
dsRNA-injected eggs were allowed to undergo
parthenogenetic development. The same dsRNA wasinjected into the hemocoel of female pupae homozygous
for the GFP transgene for parental RNAi. Eclosed adults
were aged for a week and mature eggs were artificially
activated to induce parthenogenetic development. Table 1
summarizes the results of knockdown effects as evaluatedbased on the phenotype (absence of GFP fluorescence).
Although both embryonic RNAi and parental RNAiknocked down the targeting GFP transgene during
embryonic development, about one fourth of survived
embryos subjected to embryonic RNAi exhibited GFP
fluorescence at 96 h of embryogenesis. In contrast, GFP
fluorescence was not detected in all embryos until the first
instar larval stage in parental RNAi. Most eggs taken from
GFP dsRNA-injected mothers developed normally, while
about a half of GFP dsRNA-injected eggs did not survive
apparently due to the damage caused in the injection
procedure.
Similar results were obtained when the Dll gene wastargeted (Table 2). When a 350-bp-long dsRNA corresponding
to the 5ʼ region including the Dll open reading frame (ORF)
was injected into eggs taken from wild-type females(embryonic RNAi), about 60% of the Dll dsRNA-injected
eggs survived and hatched. Nearly 90% of hatched larvae
had defects in presumed Dll-expressing structures, suchas the distal region (telopodite) of appendages.

* Abstract of paper read at the 47th Annual Meeting of the Arthropodan Embryological Society of Japan, June 10–11, 2011 (Biwako, Shiga).

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Table 1 Comparison of knockdown effects targeting the GFP transgene
Percent of individuals that lost GFP fluorescence

No. of eggs
activated

No. embryos normallydeveloped at 48 h (%)

48 h embryos

96 h embryos

1st instar larvae

3rd instar larvae

Embryonic RNAi
GFP dsRNA
D.W.

355
193

200 (56.3)
105 (54.4)

100 (n=200)
0 (n=105)73.4 (n=199)
0 (n=102)40.3 (n=159)
0 (n=97)0 (n=116)0 (n=85)

Parental RNAi
GFP dsRNA
D.W.

361
338

312 (86.4)
305 (90.2)

100 (n=312)
0 (n=305)

100 (n=311)
0 (n=305)

100 (n=302)
0 (n=290)0 (n=278)0 (n=264)

Table 2Comparison of knockdown effects targeting endogenous Dll gene
Percent of individuals showing knockdown phenotype
SevereMildNone(complete loss)(partial loss)(normal)No. of eggs
activated

No. of larvae hatched andexamined (%)

Embryonic RNAi
Dll dsRNA393

239 (60.8)

46.9

41.4

11.7Parental RNAi
Dll dsRNAGFP dsRNA

344
174

305 (88.7)
162 (93.1)

1000000100

Nevertheless, the phenotype varied in degree and only
half of these defective larvae completely lost Dllexpressing structures. Contrastingly, in parental RNAi,
most eggs taken from Dll dsRNA-injected mothers
survived and all larvae showed a severe phenotype withcomplete loss in Dll-expressing structures.
Taking these results together, both embryonic RNAiand parental RNAi are effective for functional analysis ofgenes expressed during embryonic development. ParentalRNAi is more efficient and practical for gene analyses inthe sawfly considering the survival rate, less fluctuating
effects and intensity of the knockdown phenotype.
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