23Proc. Arthropod. Embryol. Soc. Jpn. 50, 23–24 (2016)Ⓒ 2016 Arthropodan Embryological Society of Japan
ISSN 1341–1527

Roles of Homeobox Genes in Proleg Development in the Sawfly,
Athalia rosae ruficornis (Hymenoptera)*
Masatsugu HATAKEYAMADivision of Insect Sciences, National Institute of Agrobiological Sciences, Owashi, Tsukuba, Ibaraki 305–8634, Japan
E-mail: sawfly@affrc.go.jp

Abdominal appendages of insects have received lessattention than thoracic legs and mouthparts, since theabsence of appendages in abdomens is one of the features ofinsects (Snodgrass, 1935; Gullan and Cranston, 2010).
Nevertheless, some holometabolous species have “unusual”
abdominal appendages in their larval stages (Matsuda, 1976;Nagy and Grbic, 1999; Bitsch, 2012). The larvae of primitive
Hymenoptera (sawflies) and Lepidoptera (moths andbutterflies) have remarkable abdominal appendages (prolegs);
however, their arrangement varies. For example, the sawfly(Athalia rosae ruficornis) has seven pairs of prolegs from thesecond abdominal segment (A2) to A8, and a pair of anal
prolegs in the last segment, A11 (Oka et al., 2010). On theother hand, the silkworm (Bombyx mori) has four pair ofprolegs in A3-A6, and anal prolegs in A11 (Scoble, 1995;
Tomita and Kikuchi, 2009). The following questions thus
arise: what is the origin of the prolegs (serial homologues of
cephalic and thoracic appendages)? and what is the underlying
molecular regulation of proleg formation? Prolegs of A. rosae
ruficornis were characterized by morphological and gene
expression analyses, and proposed that they are coxopodal
endites (outgrowths of ventral plates), but not the main shaftof appendages (Oka et al., 2010). It is considered that thehomeobox genes belonging to the Bithorax Complex (BXC),
Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B(Abd-B), are involved in the primary regulation of abdominal
appendage development (Hughes and Kaufman, 2002;
Robertson and Mahaffey, 2009). In this study, the functions ofthese three genes were analyzed by knocking down theirtranscripts during embryonic development.
Ubx, abd-A and Abd-B of A. rosae ruficornis have beencloned and characterized (Hatakeyama et al., 2013). As RNA
interference (RNAi) induced by the injection of doublestranded RNA (dsRNA) works very effectively in A. rosae
ruficornis (Yoshiyama et al., 2013), a short dsRNA corresponding
to each gene transcript was synthesized and injected into
hemocoel of the parental mother pupae. After adultemergence, eggs were taken from the dsRNA-injectedmothers and allowed to develop parthenogenetically. Thegene expression and phenotypes were examined during

embryogenesis. The transcripts of each gene were specificallyknocked down in embryos when body segments were formed
and appendage primordia appeared. The larval phenotypes
upon gene knockdown in comparison to normal phenotype
were as follows:
Normal phenotype:・No appendages were formed in A1.
・Seven pairs of prolegs were formed in A2-A8.
・No appendages were formed in A9, and in A10 which
is usually reduced with only a small dorsal arearemaining.
・A pair of anal prolegs was formed in A11.
Ubx knockdown phenotype:・A pair of basal thoracic appendage-like structuresappeared in A1, where appendages are not originallyformed.
・Seven pairs of prolegs were formed normally in A2A8.
・A pair of anal prolegs was formed normally in A11.
abd-A knockdown:
・No appendages were formed in A1.
・All prolegs in A2-A8 were absent and only rudimentary
structures remained.
・A pair of anal prolegs was formed normally in A11.
Abd-B knockdown:
・No appendages were formed in A1.
・Seven pairs of prolegs were formed normally in A2A8.
・A pair of prolegs appeared in A9, and in A10 which isformed similar in appearance to the other abdominalsegments.
・A pair of anal prolegs was formed normally in A11.
These results suggest the following functions of homeobox
genes in A. rosae ruficornis abdominal appendage
development: Ubx suppresses appendages in A1, abd-A
promotes prolegs in A2-A8, and Abd-B specifies the posterior
abdominal segments and suppresses prolegs in A9 and A10.
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