In Vivo MMP Functions Figure 6. Quantitative real time RT-PCR analysis of transcriptional levels of Tribolium MMPs during metamorphosis. (A) The mRNA levels of MMP-1 (black bars), MMP-2 (gray bars), and MMP-3 (white bars) of different metamorphosis stages is shown relative to their expression levels in last instar larvae. Expression rates of MMP-1 increased 4-fold during prepupal stage and 8-fold during early pupal stage (1–3 h) and declined soon after this to basal levels. In contrast, MMP-2 mRNA levels were determined to be elevated at all analyzed prepupal and pupal stages with the highest elevation (15-fold) at the early pupal stage when compared to larval stage. Interestingly, MMP-3 expression levels were not induced during early stages but in a later stage of metamorphosis. In 3 day old pupae, we determined a 6-fold elevated transcript level. (B) TIMP gene expression was repressed 2-fold in early metamorphosis and induced in later stages, whereas RECK gene expression was induced during all tested stages of metamorphosis. Expression levels of MMP-4 (Glean 10983) and 18S rRNA genes were not significantly different at all analyzed stages (data not shown). Results represent mean values of three independent determinations6S.D. from ten animals per stage that were pooled for analysis. doi:10.1371/journal.pone.0004751.g006 septic injury in immune-competent hemocytes [36]. Therefore, we investigated here the potential role of MMPs in insect innate immunity using model host insect T. castaneum [37]. Upon septic injury we observed significantly increased mRNA levels of about 2 to 5 fold of MMP-1, TIMP, and RECK, respectively, in immune- competent Tribolium hemocytes along with the immune-inducible antifungal Tribolium thaumatin (Fig. 9A). In contrast, mRNA levels of MMP-2 and MMP-3 were not significantly influenced by the treatment. In whole animals including the fat body, which is responsible for massive immune-induced production of antimicro- bial peptides we determined only a weak and not significantly induced expression of MMP-1 (Fig. 9B). These results suggest that MMP-1 functions mainly at the cellular level of innate immunity and little in the systemic immune response. To confirm our results, we examined enzymatic activities of hemocytes and observed MMP-like enzymatic activity localized at the surface of stimulated hemocytes, which lead to the degradation of both fluorescently labeled collagen IV and MMP-1/MMP-9 peptidic substrate (Fig. 10A,B). Knock-down of MMP-1 significantly reduces survival time of infected beetles To obtain direct evidence that MMPs are involved in insect innate immunity, we knocked-down MMPs, TIMP, and RECK, respectively, in T. castaneum imagoes. Subsequently, three days later we infected them with the fungal entomopathogen Beauveria bassiana by injecting conidia spores into the hemocoel. Survival time of infected larvae was determined by counting live and died animals for several days upon infection. MMP-1 (RNAi) beetles died significantly faster than control RNAi animals, indicating an essential role of MMP-1 in proper insect innate immunity (Fig. 11). In contrast, knock-down of MMP-2, MMP-3, MMP-4, TIMP, and RECK, respectively, resulted in no altered susceptibility of beetles to B. bassiana (data not shown). Purification of recombinant Tribolium MMP-1 and determination of its collagenolytic activity To investigate the enzymatic properties of MMP-1, we produced recombinant Tribolium MMP-1 in E. coli. Tribolium MMP-1 was purified from the urea-soluble E. coli inclusion bodies fraction by immobilized metal affinity chromatography (Fig. 12A). Auto-activation of purified and refolded protein resulted in active MMP-1 which was able to degrade collagen IV in vitro (Fig. 12B). Under examined conditions the reaction velocity of one mg recombinant Tribolium MMP-1 was 165648 ng DQTM-collagen type IV per hour which was abolished in the presence of the specific MMP inhibitor GM6001 (Fig 12B). For comparison, using the same experimental conditions, we measured the reaction velocity of one mg recombinant human MMP-19 that was kindly provided by Radislav Sedlacek (Prague, Czech Republic) and found that it was in a similar range as that observed for recombinant Tribolium MMP-1 (data not shown). Discussion Our results reveal that the in vivo functions of MMPs in the model beetle T. castaneum are wide ranging. Use of dsRNA- meditated gene silencing revealed that MMPs play essential roles during embryogenesis as well as metamorphosis. Furthermore, our results provide the first direct evidence that MMPs are involved in regulating innate immunity in insects. In Tribolium, dsRNA-mediated genetic interference has been used recently to unravel novel functions of developmental transcription factors and morphogens in insect embryogenesis and metamorphosis [24]. Some of the regulative processes have been demonstrated to be similar, whereas others to be strikingly different to processes known from Drosophila. For example, in Tribolium, patterning of its dorsoventral axis is controlled by homologous genes that are also used in Drosophila, but rather than relying on maternal information, PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4751