Functional Characterization of a Juvenile Hormone Esterase Related Gene in the Moth <i>Sesamia nonagrioides</i> through RNA Interference

<div><p>Juvenile hormone esterase (JHE) is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (<i>SnJHER</i>) in the corn stalk borer, <i>Sesamia nonagrioides</i>. <i>SnJHER</i> was rhythmically up-regulated close to each molt during the corn stalk borer’s larval development. In this paper we attempted to functionally characterize <i>SnJHER</i> using several reverse genetics techniques. To functionally characterize <i>SnJHER</i>, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi). Our findings indicate the potential implication of <i>SnJHER</i> in the developmental programming of <i>Sesamia nonagrioides</i>. It is still unclear whether <i>SnJHER</i> is closely related to the authentic <i>JHE</i> gene, with different or similar biological functions.</p></div

Targeting the <i>SnJHER</i> 472 bp part after baculovirus administration of dsJHER₄₇₂.

<p>Developmental abnormalities of pupal-adult transition of BmNPV-BmA::GFP/BmA::dsLuciferase and BmNPV-BmA::GFP/BmA::JHER_loop infected animals. The scale less phenotype was presented only in the BmNPV-BmA::GFP/BmA::JHER_loop infected animals. Scale bar: 1 cm</p

Targeting the <i>SnJHER</i> 472 bp part after baculovirus administration of dsJHER₄₇₂.

<p>% emerged adults from pupae infected with the BmNPV-BmA::GFP/BmA::dsLuciferase and BmNPV-BmA::GFP/BmA::JHER_loop viruses infected as L5d3 or L6d9 larvae. The experiment was replicated 3 times and the mean and the statistical significance of the percentage of the emerged adults between the control and the experimental group (p<0.05, t-test) were calculated.</p

Summary of the results after hemolymph, bacterial, baculovirus-mediated dsJHER administration.

<p>The asterisk indicates the total amount of insects used, among three independent trials, both for control and experimental groups.</p

Targeting the <i>SnJHER</i> 472 bp part after baculovirus administration of dsJHER₄₇₂.

<p><b>A.</b> Fluorescence field images of BmNPV-BmA::GFP/BmA::dsLuciferase and BmNPV-BmA::GFP/BmA::JHER_loop infected animals. <b>B.</b> Semiquantitative RT-PCR analysis of <i>SnJHER</i>, JHER_loop and <i>GFP</i> gene in randomly selected BmA::GFP/BmA::dsLuciferase (1) and BmNPV-BmA::GFP/BmA::JHER_loop infected animals (2), 7 days post infection <b>C.</b> Real time RT-PCR analysis of <i>SnJHER</i> mRNA levels in 14 randomly selected BmA::GFP/BmA::dsLuciferase (White column) and BmNPV-BmA::GFP/BmA::JHER_loop (Black column) infected individuals, 7 days post infection. The bars above the columns indicate the S.E. of the mean of 14 samples with three technical replicates.</p

Targeting the <i>SnJHER</i> 472/1276/1725 bp part after hemolymph injection of L5d3 and L6d9 larvae.

<p><b>A.</b> Schematic representation of <i>SnJHER</i> gene. The black color represents the <i>SnJHER</i> ORF, while the white color represents the 5′ and 3′ untranslated regions of the gene. <b>B., E.</b> Semiquantitative RT-PCR analysis of <i>SnJHER</i> mRNA levels, of a randomly selected individual injected with the dsJHER₄₇₂ as 5^th instar d3 (B.) or 6^th instar d9 larva (E.) and its randomly selected control 3 days post injection. <b>C., F.</b> Semiquantitative RT-PCR analysis of <i>SnJHER</i> mRNA levels, of a randomly selected individual injected with the dsJHER₁₂₇₆ as 5^th instar d3 (C.) or 6^th instar d9 larva (F.) and its randomly selected control 3 days post injection. <b>D., G.</b> Semiquantitative RT-PCR analysis of <i>SnJHER</i> mRNA levels, of a randomly selected individual injected with the dsJHER₁₇₂₅ as 5^th instar d3 (D.) or 6^th instar d9 larva (G.) and its randomly selected control 3 days post injection.</p

Primers used in this study.

<p>Primers used in this study.</p

Targeting the <i>SnJHER</i> 472 bp part after bacterial administration of dsJHER₄₇₂.

<p><b>A.</b> Confirmation of dsJHER₄₇₂ synthesis in IPTG induced HT115 bacteria. Agarose gel electrophoresis of RNA extracted from IPTG induced HT115/L4440 (1) and HT115/pGEM T-easy-JHER_loop (2) bacteria followed by RNase-A treatment in high salinity buffer. <b>B.</b> Semiquantitative RT-PCR analysis of JHER gene in randomly selected pools of 10 insects recovered from a continuous feeding assay (1^st→5^h instar d0) with the HT115 bacteria (1, 2), 6 and 15 days post recovery (PR). <b>C.</b> Semiquantitative RT-PCR analysis of <i>JHER</i>, <i>EcR</i>, <i>Hsp70</i> and <i>Hsc70</i> in randomly selected pools of 10 insects from a continuous feeding assay (5^th→6^th instar) with the HT115 bacteria (1, 2), 7 days post feeding. As reference gene it was used the <i>Sesamia</i>’s <i>b-tubulin</i> gene.</p