initiation of ecdysis, the larvae were unable to shed their puparium. After removal of the apolysed larval cuticle the treated animals looked like pupal-larval intermediates with elongated body shape and untanned cuticle (Fig. 3C,D). Further examination revealed distinct visible abnormalities in some structures. The compound eye, for example, developed several rows of ommatidia but in abnormal arrangement when compared to wild-type pupae (Fig. 4I). The thorax of MMP-1 (RNAi) animals developed a pupa-like, not fully developed pronotum and the dorsal abdomen revealed a line of short setae, cuticular structures observed in the pupal stage (Fig. 4A,C). The wings were rudimentary (Fig. 4A,Y) but other characteristics like gin traps (Fig. 4U), genital papillae, and urogomphi were well developed (Fig. 4ZC). Mouthparts (Fig. 4M) were nearly fully developed including a pupa-like labrum and separated mandibles with a gap between the incisors (Fig. 4M). The antennae were thick (Fig. 4E), the head antrorsed (Fig. 4I), and the legs short and distant though they did possess the double claws that normally develop in pupae and are typical for the adult leg (Fig. 4Q). When MMP-1 dsRNA was injected into pupae no distinct phenotype appeared and individuals developed to normal adults exhibiting normal egg production, suggesting no importance during the pupa to imago transition. Interestingly, the fourth instar larvae injected with MMP-1 dsRNA developed and molted normally into the final instar larval stage and reached the prepupal stage, suggesting no essential involvement of MMP-1 in ecdysis between larval stages. However, achieving the prepupal stage they then arrested and showed the same lethal phenotype as described above. Using Pig-19 strain beetles, which express EGFP in muscle tissues [29] we examined muscle development. Control larvae exhibited well developed muscle fluorescence in the head and in the whole body (Fig. 5A) whereas in control pupae, fluorescence of muscles was more concentrated at the abdomen and organized in parallel strands (Fig. 5B). Observation of MMP-1 (RNAi) phenotype revealed that muscles had been mainly remodeled to pupal fate but without achieving final developmental stage (Fig. 5C). To determine at which stages of pupal morphogenesis genes of MMPs, TIMP, and RECK are expressed we analyzed RNA from final instar larvae, prepupae (pharate pupae), pupae of different ages, and imagoes by quantitative real-time RT-PCR. Our analysis revealed enhanced mRNA levels of MMP-1 and MMP-2 in early In Vivo MMP Functions pupae when compared with corresponding samples from last instar larvae, whereas MMP-3 showed an increased transcriptional rate in 3-day old pupae (Fig. 6A). Expression rates of MMP-1 increased 4- fold from larval to prepupal stage and 8-fold to early pupal stage, whereas MMP-2 mRNA levels were determined to be elevated during whole metamorphosis with highest values at early pupal stage. When compared with mRNA levels from last instar larvae, RECK was induced about 3-fold during all analyzed stages of metamorphosis, whereas TIMP was 2-fold repressed in early pupal stages similar as recently described in Drosophila [30–31] and increased up to 2-fold in later pupal stages (Fig. 6B). Expression levels of the virus-derived MMP-4 and 18S rRNA genes were not significantly different at all analyzed stages. Examination of potential regulation of TGF-b-like Decapentaplegic (Dpp) by MMP activity Page-McCaw et al. [22] noted that Drosophila MMP-1 mutant phenotype resembles the phenotype derived by mutation of Decapentaplegic (Dpp) supporting the hypothesis that MMPs may modulate the morphogen gradient directly or indirectly by degrading extracellular matrix proteins. Dpp is a bone morpho- genetic protein (BMP)-type ligand that belongs to the TGF-b family with essential role during insect development. The Dpp gradient is regulated by a number of modulators that either bind Dpp, cleave Dpp-binding proteins or compete with Dpp for receptor-binding sites [25]. To investigate possible regulation of Dpp by MMP activity, we silenced Dpp expression in last instar Tribolium larvae and compared the resulting phenotype with that of MMP-1 (RNAi) phenotype. Recently, the essential role of Dpp during beetle embyrogenesis has been investigated [32] but to our knowledge this is the first study showing impact of Dpp on Tribolium metamorphosis. Efficiency of RNAi mediated Dpp knock-down was confirmed by RT-PCR analyses (data not shown). Interest- ingly, Dpp knock-down did not affect ecdysis between larval stages and larval to pupal transformation, but frequently resulted in an arrest of the pupal to adult transition leading to a phenotype with pupal morphology and with adult-like tanning of the cuticle (Fig. 7 A,B). Though this was the predominant phenotype, some animals exhibited a milder phenotype in which they ecdysed to imagoes but had affected inflated wings and leg deformations, mainly of the Figure 3. RNAi (MMP-1) knock-down Tribolium larvae arrest during larval to pupal transformation. Lateral views of whole bodies of wild- type larva (A) and arrested MMP-1 knock-down animals at the larval-pupal transformation (B) are shown. For comparison wild-type pupa (C) and MMP-1 knock-down animals that were removed from the puparium (D) are also shown. Scale bar, 1 mm. doi:10.1371/journal.pone.0004751.g003 PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4751