428 analysis of the Oncopeltus gene confidently places it as a homolog of the Drosophila D-Sp1, Tribolium Sp8, and the murine Sp8 and Sp9 genes, the exact orthology within this group is unclear and we therefore designate the Oncopeltus gene as Sp8/9. We have studied the role of the Oncopeltus Sp8/9 ortholog in order to investigate its evolutionary conservation in insect appendage development. We find that Sp8/9 is expressed in the appendages throughout development and the downregulation of Sp8/9 expression via RNAi leads to shortened legs, rostrum, and antennae. These data show that Sp8/9 is involved in the allometric growth of the appendages. However, we find no evidence for a more global role of Sp8/9 in appendage specification. Materials and methods Animal husbandry and embryology Milkweed bugs were reared as described previously (Hughes and Kaufman 2000). Collected eggs were kept at 25°C. Embryos of all stages were fixed as reported in Liu and Kaufman (2004a). Oncopeltus embryo dissections before in situ staining were performed under a fluorescence stereomicroscope using SYTOX Green nucleic acid stain (Invitrogen) (Liu and Kaufman 2004b). Isolation of Sp8/9 and sequence analysis Oncopeltus embryos from 0 to 96 h were used for mRNA isolation using the MicroPoly(A)Purist kit (Ambion). This mRNA served as template for double-stranded (ds) cDNA synthesis (SMART PCR cDNA Synthesis kit, Clontech) and RACE template synthesis (SMART RACE cDNA Amplification Kit, Clontech). PCR with the primer pair Fw_GGC MGG GCI ACI TGY GAY TGY CCI AAY TG (GRATCDCPNC) and Rev_ARR TGR TCI SWI CKC ATR AAI CKY AA (LHDSRMFRK) resulted in a fragment of 311 bp. PCR fragments were cloned into the pCRII vector (Invitrogen). Additional sequence information was obtained by 5′ RACE PCR using the reverse primer CAG GTG AGC CTT GAG GTG CGA GGT C. Phylogenetic analysis of different Sp factor sequences was performed as described previously (Prpic et al. 2005). The Oncopeltus Sp8/9 sequence is available from the EMBL nucleotide database under the accession number FN396612. In situ hybridization The longest 5′ RACE fragment of Sp8/9 (1,078 bp comprising 181 bp 5′UTR and 897 bp ORF) served as template for the synthesis of digoxygenin-labeled RNA Dev Genes Evol (2009) 219:427–435 probes (Roche). In situ hybridization was performed as described previously (Liu and Kaufman 2004a). Parental RNA interference The template for dsRNA synthesis was prepared by PCR with T7 (GAA TTG TAA TAC GAC TCA CTA TAG G) and Sp6-T7 (TAA TAC GAC TCA CTA TAG GAT TTA GGT GAC ACT ATA GA) primers from the longest 5′ RACE fragment of Sp8/9 that has also been used for probe generation. Double-stranded RNA (dsRNA) was generated using the MEGAscript T7 Kit (Ambion) and resuspended in 1× injection buffer (1.4 mM NaCl, 0.07 mM Na2HPO4, 0.03 mM KH2PO4, 4 mM KCl) at a concentration of 4 µg/µl. RNA injections in adult virgin Oncopeltus females were performed as described previously (Liu and Kaufman 2004a). Injection of 1× injection buffer served as the negative control. To verify the RNAi phenotypes obtained with the full fragment, we repeated the parental RNAi with two shorter non-overlapping fragments of Of Sp8/9. A different 5′ RACE Of Sp8/9 fragment of 1,057 bp (133 bp 5′ UTR, 924 bp ORF) was cut with XhoI which resulted in two fragments of 421 and 636 bp. Parental RNAi experiments with dsRNA transcribed from these two fragments resulted in the same phenotype with a similar frequency as for dsRNA injections with the full longest 5′ RACE fragment (data not shown). As independent RNAi controls, we performed injections of dsRNA of EGFP and Of eve which resulted in no abnormal phenotype or the same phenotypes as previously published for Of eve, respectively (Liu and Kaufman 2005) (data not shown). Results and discussion Isolation of the Sp8/9 homolog of O. fasciatus A short fragment of the Oncopeltus homolog of the Sp8/9 gene was isolated by PCR. More sequence information was obtained by 5′ RACE PCR. The available sequence comprises 181 bp of 5′ UTR followed by 1,008 bp of protein coding sequence also encoding the Btd box motif and the zinc finger motifs ZF 1, ZF 2, and a portion of ZF 3. An alignment of these conserved domains with the nine Sp factors from the mouse, as well as D-Sp1 and Btd from Drosophila and Tc Sp8 and Tc Btd (Schinko et al. 2008) from Tribolium, shows that the Btd box motif and the available sequences of the zinc finger motifs are almost identical in all Sp8/9 proteins, but clearly different from the homologous motifs in other Sp proteins (Fig. 1a). The linker regions between these motifs are also highly conserved in all Sp8/9 proteins, whereas differences in sequence and linker length exist in other Sp proteins (Fig. 1a).