tropomyosin isoform 2, Boceropsin, DNA supercoiling factor, EN16b presenilin enhancer, calreticulin, ecdysteroid-regulated 16 kDa protein precursor, Scr, allatostatin receptor, dopamine transporter, caspase-1, allatostatin receptor Bras2, Wcp4, presenilin enhancer, Pbanr, Rack1, BIR, cell deathregulatory protein BIR, Jhamt, Iap, ecdysone receptor, allatostatin preprohormone, allatostatin receptor, Rieske-domain protein Neverland, BmCF1, Eck, thymosin isoform 2, chitinase-like 
protein BmDHR-2, cell death-regulatory protein, BMSR, serine/threonine protein phosphatase 6, PKG-II, Sgf-1, allatostatin preprohormone, dopa decarboxylase, Adamts-like protein, IPPI_Bm 3 4 5 6 Nutlin3 bmo-miR-8 bmo-miR-9a bmo-miR-10 bmo-miR-13 Ptp99A, atrophin, Br, Hr46, SOPs Scr, HOXA3 Rpr, hb, 7 8 9 10 11 12 bmo-miR-14 bmo-miR-31 bmo-miR-71 bmo-miR-77 bmo-miR-92 bmo-miR-263a Br, NetA, EcR, Eip93F, Drice NetB, grim, reaper, sickle Hr96 Abd-A, Clk, dbt,tws, slo 13 14 bmo-miR-275 bmo-miR-278 Scr, Drl-2, NetA, expanded 15 bmo-miR-iab-4-3p – Those target genes have been predicted or confirmed in Drosophila. doi:10.1371/journal.pone.0002997.t004 7 microRNAs in Silkworm metabolism at molting stage, compromising for the conflict of energy-hungry process and fasting behavior by targeting insulin receptor-like protein precursor, bombyxin, and BmCF1. Our cloning and real-time PCR results showed the expression of miRNAs is outstanding in stage of MLS, both in sorts and quantities. These discoveries shed lights 16722652 on the fine-tuning mechanism of miRNAs in regulating different developmental stages of B. mori. Further investigations are needed to expand our comprehensive understanding of the modulating networks of miRNAs in the development of B. mori and even other insects. Materials and Methods Experimental materials from silkworms Our starting materials were from HCl-treated diapaused eggs of Chinese silkworm strain Dazao provided by the Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, P. R. China. Eggs were incubated at 2561uC under illumination, and larvae were reared on an artificial diet produced by 25137254 the Sericultural Research Institute of Shandong, P. R. China. Eggs were incubated at 25uC for 30 days, and then transferred to 4uC and kept cold for 2 months to terminate diapause. All developmental stages of the silkworm were obtained in the following manner: eggs laid within a 20-hour period for pre-diapause stage, eggs laid within 40 48 hours for diapause stage, chilled eggs just before revival for diapause-broken stage, eggs collected 72 hours after diapause-broken stage for blastokinesis stage, eggs collected 120 hours after diapause-broken stage for tracheaappearing stage, eggs collected 200 hours after diapause-broken stage for bluish stage, larvae hatched at the first day were collected for newly-hatched larva stage, larvae collected on day 3 of the fourth-instar for fourth-instar larva stage, larvae collected at fourth-molting for molting larva stage, larvae collected on day 4 of the fifth-instar for late fifth-instar larva stage, larvae collected at spinning for spinning stage, larvae collected 2 day after cocooning for pre-pupa stage, pupas collected 6 days after cocooning for pupa stage, and moths collected before mating for moth stage. Computational prediction of microRNAs We used Srnaloop to predict putative miRNAs from the silkworm genome. The genome assembly and some functional annotations were downloaded from SilkDB and Silkworm gen
