Expression pattern of the BmNPV <i>ie1</i>-EGFP transgene in tissues dissected from 5th instar silkworm larvae.

<p>(A, A’) A dissected non-transgenic 5th instar larva. (B, B’) A dissected transgenic 5th instar larva. Intense EGFP fluorescence was evident in the anterior and posterior midgut. (C–J’) A dissected 5th instar larva. (C) Prothoracic gland. (D) Merged image of the transmitted light and EGFP fluorescence in the suboesophageal body. (E–F’) Trichogen (or trichogen and tormogen) cells in the epidermis in a non-transgenic larva (E, E’) and a transgenic larva (F, F’). (G–H’) Ovary of a non-transgenic larva (G, G’) and a transgenic larva (H, H’). EGFP was evident in tracheolar cells that were attached to the ovary. (I–J’) Tissues surrounding dorsal vessel of a non-transgenic larva (I, I’) and a transgenic larva (J, J’). EGFP was evident in pericardial cells along dorsal vessel and on the alary muscle, and peritracheal athrocytes, but not in fatbody. (A, B, E–J) White light, (A’, B’, C, E’-J’) EGFP-excitation wavelength light. The images for the comparisons of non-transgenic and transgenic larvae and tissues were obtained exactly by the same conditions. Abbreviations: dv, dorsal vessel; fb, fatbody; mg, midgut; pa, peritracheal athrocytes. Scale bars = 5 mm in (A) and (B), 500 µm in (C, E, F, G, H), 1 mm in (I, J).</p

Schematic representation of the <i>piggyBac</i>-based BmNPV <i>ie1</i> promoter reporter constructs.

<p>This construct was designated pBac[BmNPV <i>ie1</i>-EGFP, 3xP3-DsRed]. A fragment containing the sequences –631 to –2 bp upstream of the codon encoding the translational start site of BmNPV <i>ie1</i> was used as the BmNPV <i>ie1</i> promoter and to drive expression of EGFP. DsRed was under the control of 3xP3 and was used as the transformation marker. Abbreviations: ITR, inverted terminal repeats of <i>piggyBac</i>; hsp70 polyA, hsp70 polyadenylation signal; SV40 polyA, SV40 polyadenylation signal.</p

Developmental expression profiles of <i>OtDH-PBAN</i>.

<p>A; RT-PCR analysis was performed on day 3 in sixth instar larvae (LVI3) as well as on day 1 in pupae (P1). Levels of DH-PBAN (DHP, lanes 1–13) and actin (lanes 14–26) mRNAs were examined. BS, brain-subesophageal ganglion complex; MG, midgut; FB, fat body; SL, silk gland; OD, ovarian disc; WD, wing disc; IM, integument and muscle; WG, wing; OV, ovary. Whole-mount <i>in situ</i> hybridization was performed in larval (B) and pupal (F) brain-SG complexes by using antisense RNA of <i>OtDH-PBAN</i> as a probe. Using anti-FXPRLa antibody, immunohistochemistry was performed in larvae (C) and pupae (G) stages. Magnified double stained images are shown in magenta (DH-PBAN RNA) and green (anti-FXPRLa) in larval (D) and pupal (H) SG, and larval corpus cardiacum, CC (E). Immunostaining was performed in pupal CC (I). FXPRLa immunoreactive somata were detected in three neuromeres, mandibular cells (SMd), maxillary cells (SMx), and labial cells (SLb), located along the ventral midline. The projective axons (arrowhead) from these somata run into the CC via the circumesophageal connective (CoC). Scale bar = 10 µm.</p

A Baculovirus Immediate-Early Gene, <em>ie1</em>, Promoter Drives Efficient Expression of a Transgene in Both <em>Drosophila melanogaster</em> and <em>Bombyx mori</em>

<div><p>Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, <em>ie1</em>, from the <em>Bombyx mori</em> nucleopolyhedrovirus (BmNPV) could be used to drive efficient transgene expression in a wide variety of insects. We used a <em>piggyBac</em>-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV <em>ie1</em> promoter; we performed a detailed investigation of the promoter in transgene expression pattern in <em>Drosophila melanogaster</em> and in <em>B. mori</em>. <em>Drosophila</em> and <em>Bombyx</em> belong to different insect orders (Diptera and Lepidoptera, respectively); however, and to our surprise, <em>ie1</em> promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole) in both insects. Furthermore, in both species, the <em>ie1</em> promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous <em>ie1</em> promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the <em>ie1</em> promoter can be used as an efficient expression driver in a diverse range of insect species.</p> </div

Expression pattern of the BmNPV <i>ie1</i>-EGFP transgene in silkworm larva at different stages.

<p>(A, A’) First instar larva just after hatching. BmNPV <i>ie1</i> promoter-driven EGFP expression did not overlap with 3xP3-driven DsRed expression in the ventral nerve cord. Ventral view. (B) Late 2nd instar larva. EGFP was expressed throughout the whole body and throughout all larval stages. Dorsal view. (C) Early 3rd instar larva. Dorsal view. (D, D’) 4th instar larvae. Dorsal view. Upper larva is a non-transgenic larva. (E) Head and thorax of 5th instar larva. Lateral view. Scale bars = 2 mm.</p

Expression pattern of the BmNPV <i>ie1</i>-EGFP transgene in silkworm pupa.

<p>(A–C) Two-day old pupa. EGFP expression was evident throughout the pupal body. (A, A’) Dorsal views under white light (A) and EGFP-excitation wavelength light (A’). Upper pupa is non-transgenic pupa. (B) Ventral view. (C) Lateral view. (D) A ventral view of the head and thorax of 4-day old pupa. DsRed expression was evident in the compound eyes, whereas EGFP was not. Abbreviations: an, antenna; ce, compound eye; sp, spiracle; wg, wing. Scale bar = 5 mm.</p

Seasonal polyphenism in the white spotted tussock moth, <i>Orgyia thyellina</i>.

<p><i>Orgyia</i> exhibits seasonal changes in various morphological, physiological, and behavioral traits, which are determined by the photoperiod during the late larval stages from 4^th to 5^th instar larvae. In the long-day condition (LD), the larval integument becomes light colored in the final instar larva (6^th instar) as does the pupae of the females and their cocoons. The larval integuments and cocoons are darkly colored under short-day conditions (SD). In the adult stage, LD females are flight-capable long-winged morphs, but SD females are flightless short-winged morphs. Furthermore, LD female lay non-diapause eggs, whereas the SD female lay diapause eggs, which are arrested in early embryonic development. These diapause eggs are heavier in weight, larger in size, and much thicker in the chorion than non-diapause eggs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024213#pone.0024213-Kimura1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024213#pone.0024213-Sato1" target="_blank">[4]</a>.</p

Genomic DNA sequences surrounding <i>piggyBac</i> insertions.

<p>The flanking sequences of <i>piggyBac</i> insertion in <i>D. melanogaster</i> and <i>B. mori</i> have 100% identity with the genome DNA sequences of chromosome 3L and Bm_scaf 21 in chromosome 17, respectively. Abbreviations: Dm, <i>Drosophila melanogaster</i>; Bm, <i>Bombyx mori</i>.</p

Effects of other <i>Orgyia</i> FXPRLa on diapause induction in both <i>Orgyia</i> and <i>Bombyx</i>.

<p>The <i>Orgyia</i> DH, α-, β-, and γ-SGNPs, and PBAN were injected into LD-pupae of <i>Orgyia</i> (A) and the non-diapause type of <i>Bombyx</i> (B) at various doses [3 (0), 33 (1), 333 (2), and 3333 (3) pmol/pupa], and subjected to analysis of diapause egg inducing activity. Each bar represents the mean value of 20 samples ± SD. Asterisks indicate statistically significant differences at the 5% level.</p

Schematic drawing of the DH-PBAN precursor polyprotein in <i>Orgyia</i>.

<p>A; <i>DH-PBAN</i> cDNA encoding pre-prohormone consisting of 199 amino acids. It seems to undergo post-translational processing via a series of enzymatic steps that cleave and further modify by amidation the GKR, KK, GRR, and 3 GR sequences at the C-terminal amino acid of the intermediate peptide substrates to yield the signal sequence (SS), DH, α-, β-, and γ-SGNP, and PBAN, similar to other Lepidopteran DH-PBAN precursor polyproteins. B; Alignment of <i>Orygia</i> DH-PBAN with <i>Bombyx</i> DH-PBAN. Conserved amino acids are indicated with shadow boxes; highly conserved amino acids in FXPRLa sequences are indicated with dark shadow boxes. The percentages of identical amino acids is represented on the right side of the peptide sequences. A glutamine residue at position 19 in <i>Orygia</i> DH is shown by an asterisk (*).</p