Dev Genes Evol (2008) 218:141–152 147 Fig. 5 Scanning electron micrographs of Tc-tiotsh RNAi larvae. For all panels, the scale bar represents 50um. a–c Ventral views of larval heads. Maxillary (pink) and labial (green) appendages have been shaded. d–e Lateral views with leg segments labeled as follows: coxa (C), trochanter (Tr), femur (F), tibiotarsus (Tt), and pretarsus (Pt). a Wild-type larval head showing the proper position of the maxillary (Mx) and labial (L) appendages. b Moderately affected Tc-tiotsh RNAi larva in which the labial appendages (especially the proximal region) are highly reduced. c Severely affected Tc-tiotsh RNAi larva in which the labial appendages have failed to fuse. Note that in both b and c, the ventral, posterior region of the head (presumed to derive from the labial segment) is almost completely absent. d Wild-type larva. e Tc- tiotsh RNAi larva with short legs. On each leg, the trochanter is presumably fused with the reduced coxa Tc-tiotsh is important for proper development of adult trunk segments We examined the role of Tc-tiotsh in adult development using larval RNAi (Tomoyasu and Denell 2004). The resulting adults display a number of defects in the thoracic and abdominal segments (Fig. 6). Most noticeably, these individuals have abnormalities of the T2 notum that prevent the elytra from closing properly (data not shown). We also observed mild defects in the proximal legs. In addition, adults resulting from Tc-tiotsh larval RNAi show abnor- malities in many of the ventral sclerites (sternites) (see El- Kifl 1953 for a description of wild-type structures). In the ventral prothorax (T1) of RNAi-treated individuals, the epimera, which are normally located posterior to the T1 coxae, are missing and the basisternum is reduced in size resulting in the exposure of membranous areas (Fig. 6b,d). In addition, the shape of the sternellum (the process Table 2 Tc-tiotsh RNA interference cuticular phenotypes dsRNA fragment 1358–1786 1358–1786 1848–2581 Severity of Effect None 12/74 (16.2%) 2/58 (3.4%) 0/24 (0%) Mild 4/74 (5.4%) 14/58 (24.1%) 4/24 (16.6%) Moderate 14/74 (18.9%) 39/58 (67.2%) 20/24 (83.3%) Severe 44/74 (59.5%) 3/58 (5.2%) 0/24 (0%) All results were obtained by injection of dsRNA into eggs. dsRNA fragment names refer to the nucleotides of GenBank sequence AF356647 included in the fragment. Severity of effect was classified as follows. Larvae with mild effects had short legs resulting from reduction and fusion of proximal segments. Moderately affected larvae had reductions in the ventral head and labial appendages in addition to short legs. Severely affected larvae were distinguished from those with moderate effects by the failure of the labial appendages to fuse at the ventral midline.