148 Fig. 6 Adult phenotypes resulting from Tc-tiotsh larval RNAi. All images are ventral views with anterior to the top. a Wild-type adult head and thorax. b Head and thorax of Tc-tiotsh RNAi individual. The T1 basisternum (Bs) is smaller than normal. c Enlarged view of region outlined in A. d Enlarged view of region outlined in B. The T1 epimera (Epm) and the T3 meso-spinasternum (Ss) are reduced or missing. e Wild-type adult abdomen. f Abdomen of Tc-tiotsh RNAi adult. The third abdominal segment (A3) lacks its anterior process, and an additional heavily sclerotized segment is present anterior to A3 extending between the T1 legs) is abnormal. Ventral T2 is not noticeably affected, but the T3 sternite is missing its anterior process (meso-spinasternum) (Fig. 6d). The ante- rior abdominal segments are also affected by Tc-tiotsh larval RNAi. In wild-type adults, the first and second abdominal segments (A1-A2) form an internal portion of the T3 leg sockets and are only lightly sclerotized, while A3 is distinguished by a sharply pointed process extending between the T3 legs (Fig. 6e). In Tc-tiotsh RNAi individ- uals, an additional anterior region (A1 and/or A2) is heavily sclerotized and A3 lacks its anterior process, thus resem- Dev Genes Evol (2008) 218:141–152 bling the more posterior abdominal segments (A4–A6) (Fig. 6f). Discussion We have identified and cloned the lone member of the tsh gene family from Tribolium castaneum. Our analysis of this gene provides important insights into the evolution of the tsh gene family at the levels of both sequence and function. Evolution of tsh family genes Searches of several non-drosophilid insect genome sequen- ces identified only a single tsh family member in each genome. These results corroborate previous suggestions (based on more limited data) that most insects have only a single tsh-related gene and that this is the ancestral state (Herke et al. 2005; Laugier et al. 2005). A key implication of this model is that the Drosophila genes tio and tsh are the result of a recent duplication event that occurred sometime after the divergence of mosquitoes and flies. Since that duplication, tsh has apparently diverged from the ancestral sequence faster than has tio (although both have diverged more than the Tribolium and Anopheles homo- logs). More rapid divergence by one member of a recently duplicated gene pair is not unprecedented. For example, the Hox3 paralogs zerknüllt (zen) and bicoid (bcd) are believed to have arisen by duplication late in the dipteran lineage (Brown et al. 2001; Stauber et al. 1999, 2002), but the sequence of Zen is more similar to that of the Hox 3 proteins of other insects than it is to Bcd. Much of the sequence conservation between Tsh family members is found in the zinc finger regions. However, a few additional conserved motifs are present, particularly at the protein termini. The C-terminal region is well conserved between Tc-TioTsh, Ag-TioTsh, and Tio, but this conserved region is not found in Tsh. The conservation of the N- terminal region is particularly interesting. The Tsh family members for which N-terminal sequence is available fall into two distinct classes (Fig. 2c). Ag-TioTsh and both Drosophila proteins share an extended region of identity that is completely different than the short motif (MPRRKQ) shared by the Tribolium and vertebrate proteins. This pattern of conservation suggests that an ancestral Tsh family member had the MPRRKQ motif, which was replaced with a different motif sometime before the separation of Anopheles and Drosophila within the dipteran lineage. It is not clear what the function of these disparate motifs might be, as the vertebrate Tsh proteins function very similarly to Dm-Tsh when expressed in Drosophila (Manfroid et al. 2004). Perhaps these motifs are important in a particular tissue or at a different stage in development