In Vivo MMP Functions Figure 4. MMP-1 knock-down phenotype. MMP-1 knock-down animals were analyzed in detail in comparison to wild-type larva, pupa and imago. (A–D) MMP-1 knock-down animals had early pupa-like dorsal features including thorax segment 1 (T1), wings (asterisks) and a pupa-specific abdominal line of setae (arrows). (E–H) Their antennae were thick and un-segmented (tips of antennae are indicated by arrows). (I–L) MMP-1 (RNAi) larvae displayed premature compound eyes (pupal ommatidia) and exhibited reduced larval stemmata (arrows). (M–P) The mouth of MMP-1 (RNAi) phenotype animals exhibited a pupa-like morphology; mandible (Mn), maxilla (Mx), labrum (Lb), labium (Lm), gap between mandibles (indicated by arrow lines in pupa and MMP-1 knock-down phenotype), interlocking of mandibles (indicated by asterisks in larva and imago). (Q–T) MMP-1 (RNAi) larvae had shortened legs with a double inchoate claw similar to that observed in pupa (tip of legs are indicated by arrows). (U–X) Gin-traps were present in MMP-1 knock-down animals and pupae but were absent in wild-type larvae and imagoes. (Y–Zb) In MMP-1 (RNAi) animals wings were rudimentally developed (asterisks) (Zc–Zf), but genital papillae (asterisks, covered in adults) developed into those of pupae, replacing larval pygopods (arrow-head). Furthermore, urogomphi were elongated and pupa-like (arrows, not present in imago). Scale bars: A–D, 500 mm; E–H and M– T, 100 mm; I–L and Y–Zf, 200 mm; U–X, 50 mm. doi:10.1371/journal.pone.0004751.g004 tarsus (Fig. 7 C–F), suggesting role in distal growth and differentiation of appendages. Since the Tribolium Dpp (RNAi) phenotype is strikingly different to MMP-1 (RNAi) phenotype our results indicate no or only little impact of Dpp on observed abnormalities of MMP-1 (RNAi) Tribolium beetles. Role of MMPs during embryonic development: abnormal tracheal growth by MMP-1 knock-down and gut development by MMP-2 knock-down The offspring of imagoes that were injected with MMP-1 dsRNA hatched normally and showed no external defects. However, closer examinations revealed that they display abnormal tracheal development resulting in small breaks of lateral tracheal trunks between two or three segments at the posterior end of larva (data not shown). Despite these tracheal defects the animals showed no changes in their further development and emerged normally to imagoes. Injection of dsRNA of MMP-2, MMP-3, and MMP-4, respectively, into last instar larvae or pupae had no effects on further development and morphology of imagoes. They exhibited a regular egg production and their offspring emerged at expected rates. While the offspring of MMP-3 (RNAi) or MMP-4 (RNAi) animals developed normally to adults, the first instar larvae of MMP-2 (RNAi) animals showed a distinct wandering behavior and died after few days. Closer examination revealed defects in their gut development. The hind gut of wild-type Tribolium is wound up in a typical way; the forward expanding ileum is transferred into the posterior turning colon and rectum (Fig. 8A). In contrast, the offspring of MMP-2 (RNAi) animals showed a deviant coiled hind gut; the turn of the ileum, which extends to three segments in control larvae, was half reduced and both the ileum and the colon were contorted and twisted with each other (Fig. 8B). Microscopic examination of dissected guts revealed no obvious differences between hind guts from MMP-2 (RNAi) and control (RNAi) larvae except a kink at the end of colon suggesting influence of MMP-2 on specific molecular guidance cues of gut organogenesis (Fig. 8CD). MMP activity and gene expression is induced in stimulated Tribolium hemocytes Insect hemocytes are highly motile macrophage-like cells fulfilling immune-related functions including phagocytosis, nodu- lation, and hemolymph coagulation [33–35]. Recently, we have identified a lepidopteran MMP which is induced in response to Figure 5. Muscle development in MMP-1 (RNAi) animals. (A) Pig-19 larva showed muscle specific EGFP-fluorescence throughout the whole body, with a notably strong expression at its lateral sides. (B) In control (RNAi) Pig-19 pupa the muscle EGFP-fluorescence was predominantly localized in the abdomen and visible as parallel strands. (C) MMP-1 (RNAi) larva showed a pupa-like localization of EGFP-fluorescence of muscles in the abdomen with additionally significant fluorescence in the thorax. Scale bar, 1 mm. doi:10.1371/journal.pone.0004751.g005 PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4751