is orthologous to MMP-2 from Drosophila and Anopheles and, interestingly, Tribolium MMP-3 (XP_972146) groups together with MMP-3 from Anopheles (Fig. 1A). A corresponding MMP-3 ortholog is absent in the Drosophila genome, suggesting its loss during fly evolution. Insect MMP-2 and insect MMP-3 clade with human membrane-anchored MMPs. Furthermore, we identified ten Tribolium specific MMP sequences (Glean 10983, Glean 00226, Glean 06957, Glean 09406, Glean 10302, Glean 03344, Glean 06957, Glean 01812, Glean 16133, and Glean 04281) that are most closely related to a MMP from the entomopathogenetic dsDNA virus Heliothis zea virus 1 with yet unknown function. Interestingly, similar genes are also present in the genomes of other entomopathogenic viruses, e.g. Trichoplusia ni ascovirus 2c (YP_803380) and Agrotis segetum granulovirus (YP_006303), indicat- ing a yet unknown but conserved role during virus infection of insects which may include host tissue histolysis. Some of the Tribolium virus-derived genes are located on a particular chromosomes such as Glean 10983 which is located on chromosome LG 9 and are expressed at the mRNA level since we amplified a corresponding cDNA by RT-PCR. In contrast, other predicted genes are located on contigs that are yet not attributed to a specific chromosome and need sequence verifica- tion. The deduced amino acid sequences of all Tribolium MMPs include the specific catalytic domains containing the conserved motif HEXGHXXGXXHSX6M, with the three histidine residues critical for catalytic activity by binding one zinc ion involved in In Vivo MMP Functions proteolysis, the active site residue glutamic acid, and a conserved seven amino acid methionine-containing 1,4-b-turn named Met- turn [28] (Fig. 1B). Furthermore, in the Tribolium genome, we identified one TIMP (Fig. 2A) and one RECK homolog (Fig. 2B) which share significant sequence similarities to counterparts from other animals including the basal sea anemone Nematostella vectensis and the complex Mammalia. Hence, MMPs, TIMP, and RECK are evolutionarily conserved ranging from most ancient to highly derived animals. Role of MMPs during metamorphosis: abnormal differentiation of antennae, compound eyes, appendages, and head by MMP-1 knock-down To examine the functional roles of MMPs during Tribolium embryogenesis, larval growth, and metamorphosis, we injected dsRNA from Tribolium MMP-1,-2,-3, and MMP-4 (a virus-derived Tribolium MMP, Glean 10983), respectively, into larval, pupal, and adult stages and monitored their further development. Both efficiency and selectivity of each RNAi knock-down experiment were confirmed by RT-PCR analyses (data not shown). Last instar larvae or pupae injected with MMP-2, MMP-3, and MMP-4 dsRNA, respectively, showed no defects and developed to normal imagoes, like the control dsRNA treated animals. In contrast, knock-down of MMP-1 resulted in animals that arrest develop- ment and die during early metamorphosis (Fig. 3A,B). Though they showed a dorsal split in the thorax cuticula indicating Figure 2. Phylogenetic analysis of Tribolium TIMP and RECK along with homologs from other organisms. (A) Aligned TIMP and (B) RECK protein sequences from T. castaneum along with homologs from other animals, were used to generate bayesian protein trees. Tribolium TIMP and RECK (indicated by gray shading) group well with other insect orthologs. Humans and some other animals have two or more TIMP homologs. Posterior probabilities are plotted at the nodes. doi:10.1371/journal.pone.0004751.g002 PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4751