Adults of <i>Sovia</i>.

<p>(A–C) <i>S</i>. <i>lucasii lucasii</i>; (D–F, I) <i>S</i>. <i>lucasii minor</i>; (D) Holotype; (E, F, I) Paratypes; (G–H) <i>S</i>. <i>lii</i>. (A, B, D, E) Male; (C, F–I) Female. Scale bar = 1 cm.</p

NJ tree for the partial mitochondrial COI sequences from 19 individuals of <i>Sovia</i>.

<p>NJ tree for the partial mitochondrial COI sequences from 19 individuals of <i>Sovia</i>.</p

Male genitalia of <i>Sovia</i>.

<p>(A) <i>S</i>. <i>lucasii minor</i>; (B) <i>S</i>. <i>lucasii lucasii</i>. (a) left valva, inner side; (b) aedeagus, dorsal view; (c) aedeagus, lateral view.</p

List of specimens used for sequencing.

<p>List of specimens used for sequencing.</p

Distribution map of <i>Sovia lucasii minor</i> (red triangle) and <i>S</i>. <i>lucasii lucasii</i> (blue triangle) based upon a map scanned from [12].

<p>Distribution map of <i>Sovia lucasii minor</i> (red triangle) and <i>S</i>. <i>lucasii lucasii</i> (blue triangle) based upon a map scanned from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183847#pone.0183847.ref012" target="_blank">12</a>].</p

Female genitalia of <i>Sovia</i>, ventral view.

<p>(A) <i>S</i>. <i>lucasii lucasii</i>; (B) <i>S</i>. <i>lucasii minor</i>.</p

Female genitalia of <i>Sovia lii</i>, ventral view.

<p>Female genitalia of <i>Sovia lii</i>, ventral view.</p

Table1_Evaluation of reference genes for quantitative expression analysis in Mylabris sibirica (Coleoptera, Meloidae).docx

Mylabris sibirica is a hypermetamorphic insect whose adults feed on oilseed rape. However, due to a shortage of effective and appropriate endogenous references, studies on molecular functional genes in Mylabris sibirica, have been tremendously limited. In this study, ten internal reference genes (ACT, ARF1, AK, EF1α, GAPDH, α-TUB, RPL6, RPL13, RPS3 and RPS18) were tested and assessed under four selected treatments including adult ages, adult tissues, temperatures, and sex by RT-qPCR based on five methods (Ct value, geNorm, NormFinder, BestKeeper and RefFinder). Our findings showed that RPL6 and RPL13 were the most optimal internal reference gene combination for gene expression during various adult ages and under diverse temperatures; The combination of RPL6 and RPS18 was recommended to test gene transcription levels under different adult tissues. AK and RPL6 were the best reference genes in male and female adults. RPL6 and RPL13 were the most appropriate reference gene pair to estimate gene expression levels under four different tested backgrounds. The relative transcript levels of a uridine diphosphate (UDP)-N-acetylglucosamine-pyrophosphorylase (MsUAP), varied greatly according to normalization with the two most- and least-suited reference genes. This study will lay the basis for further molecular physiology and biochemistry studies in M. sibirica, such as development, reproduction, sex differentiation, cold and heat resistance.</p