ATGGTTGCAAAGCTGAAACT CGATAGGGCCTTTGCTGTGG GCGATGTGTGTGTGCTGTGG GTGGGTTGGACGGGAATGAG CGTTGTGGATTGCCTGATGC GGCAGTAACGGTGGCGTACC ACAGCTGCCGAAAAGCTTGC GGCAACGGGGTTATTGCTTG TAATACGACTCACTATAGGGAGATCCAGAGTTTTGCCGGTCTC TAATACGACTCACTATAGGGAGATTGGAAGTCCGAATCGTCGT TAATACGACTCACTATAGGGAGACGGGATTTGATGGGAATGGA TAATACGACTCACTATAGGGAGATTTGTCGTCGTCGGGGAGTT TAATACGACTCACTATAGGGAGACTCCGAAGGGTGGGGAAAAC TAATACGACTCACTATAGGGAGACGTCAAAGGAGGCGTCACAA TAATACGACTCACTATAGGGAGACGTTGTGGATTGCCTGATGCT TAATACGACTCACTATAGGGAGAGGGATTTTTGCGTTGTTGTTGTTG TAATACGACTCACTATAGGGAGATGACCGCATCTGACGAAGCA TAATACGACTCACTATAGGGAGA Reverse primer 59–39 TCCCGTGTTGAGTCAAATTA CACCCCCTGGACTCACTCGT CGTTGCACCGAATCATCGAC GGCGCCTCCTCCGCTATATT CACATGTGCACGCCACGTTA TCACGAAGTCGGCTCTGCAA GGCGTTCTAAGCAGCGGAAA ACGTGTCAGGTGTGCCGAAA In Vivo MMP Functions Table 1. Primer sequences used in the present study. Gene Forward primer 59–39 Quantitative real time RT-PCR primer RT-18S rRNA RT-MMP-1 RT-MMP-2 RT-MMP-3 RT-MMP-4 RT-TIMP RT-RECK RT-Thaumatin RNAi-MMP-1 RNAi-MMP-2 RNAi-MMP-3 RNAi-MMP-4 RNAi-TIMP RNAi-RECK Primers used for RNAi experiments PLoS ONE | www.plosone.org 12 March 2009 | Volume 4 | Issue 3 | e4751 TAATACGACTCACTATAGGGAGAATTGCCACAACCTGCCCAAA TAATACGACTCACTATAGGGAGA TCCGAACCACCGAGAAATGG TCGTTTGCACCGTCACGAAA TAATACGACTCACTATAGGGAGATCGACACTGTTGCCCCTTTT TAATACGACTCACTATAGGGAGATGGTTGGTTTTGGGGTCTTG TAATACGACTCACTATAGGGAGACGGTGGAGCCTGCGATAATG TAATACGACTCACTATAGGGAGACGACGGTGGAGGGGAGTCAA RNAi-Dpp RNAi-IMPI (control) doi:10.1371/journal.pone.0004751.t001 sapiens (Human19, Q99542; Human28, Q9H239; Human11, P24347; Human21, Q8N119; Human23, O75900; Human17, Q9ULZ9; Human25, Q9NPA2; Human14, P50281; Human15, P51511; Human16, P51512; Human24, Q9Y5R2; Human20, O60882; Human12, P39900; Human13, P45452; Human1, P03956; Human8, P22894; Human3, P08254; Human10, P09238; Human7, P09237; Human26, Q9NRE1; Human2, P08253; Human9, P14780), Heliothis zea virus 1 (AAN04364), and Hydra vulgaris MMP (AAD45804). Accession numbers of TIMPs used for sequence alignment and phylogenetic analyses are as follows: Tribolium (XP_001810132), Nematostella vectensis (TIMP1, XP_001625449; TIMP2, XP_001625448), Drosophila melanogaster (NP_524301), Anopheles gambiae (XP_001231186); Aedes aegypti (EAT34208); Crassostrea gigas (TIMP1, AAG42824; TIMP2, AAT73610); Homo sapiens (TIMP1, P01033; TIMP2, P16035; TIMP3, P35625; TIMP4, Q99727). Accession numbers of RECKs used for sequence alignment and phylogenetic analyses are as follows: Tribolium (XP_974450), Nematostella vectensis (XP_001635685), Drosophila melanogaster (NP_648733), Apis mellifera (XP_392031); Aedes aegypti (XP_001657759); Homo sapiens (NP_066934). For phylogenetic reconstruction, we used the software package MrBayes 3.1.2 [59], which combines Bayesian inference and Markov chain Monte Carlo convergence acceleration techniques known as Metropolis coupling. The best fixed-rate model of amino acid evolution was determined by model jumping among nine possible models. The model with the overall highest posterior probability was WAG [60] for MMPs, TIMPs, and RECKs. The average standard deviation of split frequencies at 106 generations was 0.0021 for MMPs, and 0.001 and 0.0018 for TIMP for RECK, respectively, at 105 generations. These values indicated that the two chains that were run in each analysis converged on similar results. The 50% majority rule tree presented here was constructed from all sampled trees with the first 25% of all trees ignored as burn in. Posterior probabilities plotted at the nodes can be interpreted as the probability that the tree or clade is correct [61]. Quantitative real time RT-PCR RNA was isolated using TriReagent isolation reagent (Molec- ular Research Centre, Cincinnati, Ohio, USA) according to the instructions of the manufacturer and quantitative real time RT- PCR were performed with the real-time PCR system Mx3000P (Stratagene, La Jolla, California, USA) using the FullVelocity SYBRH Green QRT-PCR Master Mix (Stratagene), as described previously [56]. We used appropriate primers (Table 1) along with 0.1 ng of RNA per reaction to amplify 18S rRNA and 10 ng of RNA per reaction to amplify selected Tribolium genes. Primers were selected using the primer3 software [62] and were purchased from Sigma (Taufkirchen, Germany). Imaging and scanning electron microscopy EGFP expression was observed using a fluorescence stereomi- croscope (Leica MZ 16F) equipped with a GFP Plus filter set (excitation filter: 480/40 nm, barrier filter: 510 nm). Images of cuticle and gut autofluorescence were obtained on a microscope (Zeiss Axioplan 2) using a fluorescence optic. For electron microscopy animals were fixed in 80% ethanol and dehydrated in a graded series of ethanol, critical point dried using CO2 in a Balzers CPD 030, mounted on aluminium stubs, gold coated using a sputter coater 0712 B (Balzer/Leitz), and photographed with a Philips XL 20 scanning electron microscope. Expression and purification of recombinant Tribolium MMP-1 A cDNA fragment of Tribolium MMP-1, encoding the pro- and catalytic domains, was amplified by PCR with oligonucleotides