Jumpstarter method in <i>Harmonia axyridis</i>.

<p>(A) Crossing scheme for jumpstarter method. A mutator line and a jumpstarter line are crossed (G₁). Newly hatched larvae expressing both ECFP and DsRed transformation markers in the CNS were selected from the progeny (G₂). Larvae carrying both the mutator and jumpstarter elements were heat shocked to induce <i>transposase</i> expression. Newly hatched larvae expressing only the DsRed transformation marker were selected from the progeny (G₃), and analyzed by PCR for mobilization of the mutator (i.e. integration elsewhere in the genome). Each unique insertion line was used to establish a new mutator line. (B) Schematic representation of PCR method to analyze mobilization of the original mutator element. Primer set HaG1 and PLR was used to detect the original non-mobilized mutator element. If the original mutator element is remobilized, no PCR product will be detected. Primer set HaG1-HaG3 was used as a positive control to detect the homologous, mutator element-free chromosome. (C) Red circles denote progeny with apparent remobilization events. PCR amplification with the PLR-HaG1 primer set was not detected in progeny marked with a red circle, because the original mutator element was mobilized to other genomic sites. These larvae were established as new mutator lines. Each G₂ female gave at least one newly mobilized progeny. (D) Comparison of DsRed fluorescence between original mutator line (middle) and two new mutator lines (left and right). Compare to original mutator line (middle), two new mutator line larvae show higher (left) or lower (right) expression of DsRed. (E) PCR analysis of mutator element mobilization. Newly hatched G₃ larvae strongly or weakly expressing DsRed marker compared with original mutator line were selected and subjected to PCR analysis. PCR amplification with the PLR-HaG1 primer set was not detected in progeny marked with a red circle. In this case, detection efficiency of remobilized mutator element is increased compared with random selection (see result in C). (F) An enhancer-trap line of transgenic ladybird beetles. During the generation of mobilized mutator lines using the jumpstarter method, an enhancer expressing GFP throughout the body was detected (left); a wild-type control larva is also shown on the right. Panel displays a ventral view (anterior uppermost).</p

Results of inverse PCR for confirming remobilization of mutator.

<p>We performed inverse PCR using extracted DNA from original mutator individual and G₄ individuals. Each G₄ individual was derived from G₂ individuals (#2–5, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100804#pone-0100804-g003" target="_blank">Fig. 3E</a>). Each mutator inserted to different position in each genome.</p

Establishment of Transgenic Lines for Jumpstarter Method Using a Composite Transposon Vector in the Ladybird Beetle, <i>Harmonia axyridis</i>

<div><p>In this post-genomic era, genome-wide functional analysis is indispensable. The recent development of RNA interference techniques has enabled researchers to easily analyze gene function even in non-model organisms. On the other hand, little progress has been made in the identification and functional analyses of cis-regulatory elements in non-model organisms. In order to develop experimental platform for identification and analyses of cis-regulatory elements in a non-model organism, in this case, the ladybird beetle, <i>Harmonia axyridis</i>, we established transgenic transposon-tagged lines using a novel composite vector. This vector enables the generation of two types of insertion products (jumpstarter and mutator). The jumpstarter portion carries a transposase gene, while the mutator segment carries a reporter gene for detecting enhancers. The full-composite element is flanked by functional termini (required for movement); however, the mutator region has an extra terminus making it possible for the mutator to remobilize on its own, thus leaving an immobile jumpstarter element behind. Each insertion type is stable on its own, but once crossed, jumpstarters can remobilize mutators. After crossing a jumpstarter and mutator line, all tested G₂ females gave rise to at least one new insertion line in the next generation. This jumping rate is equivalent to the P-element-mediated jumpstarter method in <i>Drosophila</i>. These established transgenic lines will offer us the ideal experimental materials for the effective screening and identification of enhancers in this species. In addition, this jumpstarter method has the potential to be as effective in other non-model insect species and thus applicable to any organism.</p></div

Schematic of composite vector and overview of genomic integration.

<p>(A) The left half of the composite vector contains a 5′ ITR, a <i>transposase</i> gene under the control of the <i>Dmhsp70</i> promoter and the 3xP3-<i>ECFP</i> transformation marker, while the right half contains both 5′ and 3′ ITRs, and is marked by both an <i>EGFP</i> gene under the control of the <i>Dmhsp27 </i>minimal promoter for enhancer trapping and a 3xP3-<i>DsRed</i> transformation marker. The <i>transposase</i> gene can be immobilized by the precise excision of the sequence between the central 5′ ITR and the 3′ ITR; in this case, larvae expressed ECFP alone. (B) Transformation markers expressed in the CNS of first instar larvae of <i>H. axyridis</i> indicate vector integration. From left to right, bright field, DsRed, ECFP and EGFP are presented in wild-type, composite vector (DsRed and ECFP are expressing), Jumpstarter (only ECFP is expressing) and Mutator (only DsRed is expressing). EGFP expression is not detected in all of strains. All panels display a ventral view (anterior uppermost).</p

Reverse transcriptase PCR (RT-PCR) analysis of <i>piggyBac transposase</i> expression.

<p>Total RNA was extracted from transgenic pupae that expressed only the ECFP marker, and from wild-type pupae reared at 25°C. A reaction without reverse transcriptase (-RT) was performed with cDNA synthesis as a negative control. <i>Harmonia axyridis ribosomal protein 49</i> (<i>Ha-rp49</i>) was used as an internal control.</p

Transformation efficiency of <i>piggyBac</i> vector.

<p>Transformation efficiency of <i>piggyBac</i> vector.</p

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List of primer sequences for dsRNA synthesis. (XLS 18 kb

Focal stag beetle <i>Cyclommatus metallifer</i>.

<p>(<b>A</b>) Intraspecific sexual dimorphism and male variation in <i>Cyclommatus metallifer</i>. Female (left), small male (center), and large male (right) are shown. Scale bar indicates 20 mm. (<b>B</b>) This species exhibits strong sexual dimorphism of mandibles between the sexes. Mandibles of female (left) and large male (right) are shown. This difference in size is a result of male-specific disproportionate mandibular growth. Scale bar indicates 10 mm. (<b>C</b>) Maxilla are not sexually dimorphic. Maxilla of female (left) and large male (right) are shown. Scale bar indicates 2 mm.</p

Effects of JHA application on <i>GFP</i>^RNAi and <i>dsx</i>^RNAi individuals.

<p>The relationships between pupal weight and mandible length were described. Sex is indicated by color of symbols (male: blue and light blue, female: pink and orange). Hormone treatments are indicated by shape of symbols (acetone treatment: circle, JHA treatment: square). RNAi treatments are distinguished by closed (<i>GFP</i> dsRNA injection) or open (<i>dsx</i> dsRNA injection). Scale bars indicate 10 mm (in males) or 5 mm (in females).</p

Developmental staging chart of prepupal development from the larval-prepupal transition to pupation.

<p>Mandibular growth (cell proliferation) is indicated on the y-axis over developmental time in days on the x-axis (hashmark = 1 day). Three distinct forms are recognized - the last instar larva, the prepupa, and the pupa. Known landmarks of mandibular proliferation in large males are indicated by the red circles and shown in diagram form below the graph <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004098#pgen.1004098-Gotoh1" target="_blank">[5]</a>. The end of the larval period and the initiation of the prepupal period is indicated by Pupal Cell Construction (PCC) and is defined as the starting point of prepupal Stage 1. Outwardly the larva does not change its morphology. Stage 1 lasts approximately 2 days until the initiation of the first Gut Purge (GP) in which the prepupa begins to transform and empties out half of its gut contents. The time the prepupa spends in the first GP is known as Stage 2. Stage 3 is a quiescent phase where the prepupa undergoes massive adult imaginal tissue proliferation but outwardly appears suspended in the first GP. Stage 4 occurs over only a few hours and begins with the second GP in which the last remnants of the gut contents are egested and the prepupa completely metamorphoses into the pupa.</p