In Vivo MMP Functions Figure 9. Quantitative real time RT-PCR analysis of transcriptional levels of Tribolium MMPs, TIMP, and RECK, along with immune- inducible thaumatin during innate immune responses. The mRNA levels of MMP-1, TIMP, and RECK were significantly induced 2 to 5-fold in hemocytes in response to immune-challenge along with the immune-inducible thaumatin gene. In contrast, in whole animal preparations containing mainly fat body tissue no significantly induced expression of tested genes could be observed except immune-inducible thaumatin, although a tendency towards MMP-1 induction was detected. 18S rRNA gene expression was not influenced by treatment, and expression levels of MMP-2 or MMP-3 were not significantly different upon immune-challenge. Results represent mean values of three independent determinations6S.D. from ten animals that were pooled for analysis. Statistically significant differences were determined using Student’s t-test and are indicated (*, p,0.05; **, p,0.01). doi:10.1371/journal.pone.0004751.g009 phenotypes of Tribolium may also be a result of altered basement membrane remodeling thereby leading to abnormal pupal tissue development and differentiation. Moreover, beside basement membrane remodeling other molecular processes such as specific cell migration and autophagic cell death processes, which are known to be regulated by MMPs in mammals, may be also involved in observed phenotypes and will be investigated in future studies. Confirming this assumption, it has been recently demonstrated in Drosophila salivary glands that the ecdysone/ EcR/USP complex induces primary response gene expression during metamorphosis, including the E74, BR-C, and E93 transcription factor genes and that these genes induce second- ary-response genes, including apoptosis regulators and MMP-1 while simultaneously repressing TIMP expression [30]. Collagen degradation has been attributed to MMPs since they were first identified in 1962 by Gross and Lapie`re in tadpole tails during metamorphosis [40]. Interestingly, we found that Tribolium harbors genes encoding at least five different collagen isoforms (XP_970497, XP_970652, XP_001811664, XP_971465, and XP_974458), whereas Drosophila have only two collagen isoforms [41], indicating that the Tribolium extracellular matrix has a much more complex composition. We found that recombinant and purified Tribolium MMP-1 is able to degrade collagens, with enzymatic activity that was in a similar range to that observed for human MMP-19. Moreover, we found that several insect species including Tribolium have three MMP paralogs, whereas Drosophila lacks one of these MMPs, insect MMP-3. The existence of a third MMP homolog in Tribolium and in other insects may directly correlate with the need for a sophistically regulated degradation of a more complex extracellular matrix. However, we found no obvious change in phenotype during metamorphosis when knocking-down MMP-2 and MMP-3. This is quite surprising giving the fact that MMP-2 and MMP-3 are highly up-regulated during metamorphosis, and Drosophila MMP-2 mutants are severally affected during the larval-pupal transition. We postulate that MMP-2 and MMP-3 have overlapping functions during metamorphosis since they are more related to each other than to MMP-1. Furthermore, one or more virus-derived MMPs may have been co-opted in Tribolium for larval tissue histolysis during metamorphosis. Embryogenesis An interesting finding is that the gut development of Tribolium embryos is affected by MMP-2 knock-down. This is in striking contrast to findings from Drosophila whose MMPs are not essentially involved in embryogenesis [22]. Since Drosophila has lost one MMP during evolution we propose that regulation of gut development by MMP-2 may reflect a more ancestral state and further investigation of role of MMPs in other insects such as Anopheles or Apis, may help to unravel this role in more detail. The tracheal defects caused by MMP-1 (RNAi) are not detrimental for development and survival of Tribolium larvae. This is in striking contrast to findings from the fruit fly since Drosophila MMP-1 mutants die by hypoxy resulting by affected tracheal trunks. A possible explanation is that the more ancient tracheal system of beetles, which harbor functional spiracles ( = stigmata) at all body segments helps to survive tracheal trunk breaks, whereas Drosophila larvae which are particularly adapted to their ecological niche and have therefore reduced most spiracles [42] except anterior and posterior ones die by suffocation. PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4751