survival, angiogenesis, cell invasion, epithelial-to-mesenchymal transition, and immune surveillance [14]. In spite of their intensive investigation in vitro and in cell culture, physiological in vivo roles of MMPs are not well elucidated yet. Hence, to unravel specific underlying molecular mechanisms more research on feasible animal model organisms are urgently needed. For example, pharmacological inhibitors of MMP activity have been shown to profoundly affect development and homeostasis in species ranging from cnidarians to mammals [15,16]. In mice, knock-out mutants indicate a redundancy among MMPs and only few MMPs have been found to be required specifically during bone and vascular remodeling and mammary development [2,6]. Zebrafish lacking specific MMPs develop severe morphological abnormalities during embryogenesis including abnormal somito- genesis, organ development, and tissue architecture and are not viable [17–19]. In the fruit fly D. melanogaster, two MMPs have been identified [20,21], which control larval tracheal growth and events of pupal morphogenesis [22]. This pioneering study generated for the first time mutant organisms which were completely depleted for MMP activity and provided deeper understanding of in vivo roles of individual MMP in the fly development. Additionally, a recent study indicated that both Drosophila MMPs modulate the responses of embryonic motor axons of defined neuronal populations to specific guidance cues [23], indicating suitability of insect model organism to elucidate novel MMP functions. To better understand specific in vivo functions of MMPs, here we study their roles in the red flour beetle Tribolium castaneum. Tribolium has emerged as feasible model organism in insect evolutionary and developmental research because its complete genomic sequence is available [24] and it exhibits a more ancestral development when compared to Drosophila [25]. In this study, we identified orthologs of the two MMPs known from Drosophila, as well as a third MMP homolog, which we named MMP-3. This MMP-3 is present in Tribolium and, for example, in Anopheles but is absent in Drosophila, suggesting it may have been lost in Drosophila. In addition, we identified ten probably virus-derived MMPs in Tribolium with yet unknown functions. Taking advantage of the amenability of Tribolium for systemic and potent RNAi-mediated gene silencing [26], we knocked-down MMPs, TIMP, and RECK and observed phenotypes indicating MMP functions particularly during meta- morphosis and embryogenic organogenesis of trachea and intestine. Furthermore, knock-down of MMP-1 resulted in animals significantly more susceptible to infection by the entomopatho- genic fungus Beauveria bassiana than control (RNAi) animals providing evidence for a direct role of MMP-1 in insect innate immunity. This particular insect MMP is most related to mammalian MMP-19 whose functions in cutaneous immune response have recently been demonstrated [27]. Results provide evidence of the evolutionarily conserved and distinct roles of MMPs and their physiological inhibitors in both metazoan development and innate immunity. Results MMPs, TIMP, and RECK are evolutionarily conserved BLAST searches of the complete Tribolium genome using human and Drosophila MMP sequences resulted in the identification of three Metazoa-like and ten probably virus-derived MMP genes. Phylogenetic analysis of Tribolium MMPs together with 22 MMPs from humans, two from Drosophila melanogaster, three from Anopheles gambiae, and one from the basal animal Hydra vulgaris revealed that Tribolium MMP-1 (XP_968822) is orthologous to MMP-1 from Drosophila and Anopheles, which are most closely-related to human MMP-19 and MMP-28 (Fig. 1A). Tribolium MMP-2 (XP_969495) In Vivo MMP Functions Figure 1. Phylogenetic analysis of Tribolium MMPs along with MMPs from other organisms. A Bayesian protein tree, with posterior probabilities, was generated using aligned MMP sequences from T. castaneum, Anopheles gambiae, Drosophila melanogaster, Homo sapiens, and Heliothis zea virus 1. A Hydra vulgaris MMP was used as outgroup. This analysis revealed that Tribolium MMP-1 is orthologous to other insect MMP-1s and Tribolium MMP-2 to MMP-2 from Anopheles and Drosophila. Interestingly, Tribolium MMP-3 clades with MMP-3 from Anopheles, but a MMP-3 orthologue is obviously absent in Drosophila. Insect MMP-1 proteins show highest relation to human MMP-19 and MMP-28 whereas the other insect MMPs group together with human membrane-anchored MMPs (MMP-14 to MMP-17, MMP-24 and MMP- 25). In contrast to Anopheles and Drosophila, Tribolium has numerous genes that are most closely related to a MMP from Heliothis zea virus 1. The scale bar represents the substitutions per site according to the model of amino acid evolution applied. (B) Three Tribolium MMPs and virus-derived Tribolium MMP proteins have catalytic domains with conserved MMP specific active site sequences (MMP active site consensus sequence: HEXGHXXGXXHSX6M). For better overview sequences of only four virus-derived MMPs are shown. doi:10.1371/journal.pone.0004751.g001 PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4751