The UPLC data of juvenile hormones of various heteropteran species

Juvenile hormone (JH) plays important roles in almost every aspect of insect development and reproduction. JHs are a group of acyclic sesquiterpenoids, and their farnesol backbone has been chemically modified to generate a homologous series of hormones in some insect lineages. JH III (epoxidised methyl farnesoate) is the most common JH in insects, but Lepidoptera (butterflies and moths) and 'higher' Diptera (suborder: Brachycera; flies) have developed their own unique JH. Although JH was first proposed in the hemipteran suborder Heteroptera (true bugs), the chemical identity of the heteropteran JH was only recently determined. Furthermore, recent studies revealed the presence of a novel JH, JH III skipped bisepoxide (JHSB3), in some heteropterans, but its taxonomic distribution remains largely unknown. In the present study, we investigated JHSB3 production in 31 heteropteran species, covering almost all heteropteran lineages, through ultra-performance liquid chromatography coupled with tandem mass spectrometry. We found that all of the focal species produced JHSB3, indicating that JHSB3 is wide-spread in heteropteran bugs and the evolutionary occurrence of JHSB3 ascends to the common ancestor of Heteroptera.

Methods
For further explanation and references, see the main text of our article (Matsumoto et al., 2021. Royal Society Open Science).

Insects were collected from the field in Osaka, Nara, Kyoto, and Okayama prefectures in Japan or obtained from colonies in our and other laboratories (see, Table S1 and Figure S1). Only adults were used. Due to the limited number of individuals collected from the field, results from 14 species are based on a single specimen. Insects were individually anaesthetized on ice and immobilized by clay. The CA attached with the corpora cardiaca was removed from these individuals according to the methods outlined in a previous study [25]. In brief, the CA was incubated in 30–50 µL of the modified minimal essential medium at 30 °C for 5 h. The JHs were extracted with hexane, dried under the stream of argon gas, and dissolved again in 30-1200 µL of methanol (Table S1).

The UPLC-MS/MS (ACQUITY UPLC H-Class, Xevo TQ-S micro, Waters, Milford, MA, USA) with a chiral column (CHIRALPAK IA-U, 3.0 mm x 100 mm, 1.6 µm particle size, Daicel, Tokyo, Japan) was used to compare the retention times of JHSB3 [18,19]. Authentic JHSB3 and 10S-JHSB3 were synthesised as described previously [15]. The MS/MS analysis of the authentic JHSB3 showed the [M+H]+ ion at m/z 283.2 and the [M+Na]+ at m/z 305.3. The product ions were detected at m/z 42.9 and m/z 233.2 when ions at m/z 283.2 were used as a precursor, whereas no fragmentation was detected when ions at m/z 305.3 were used. In the present study, ions at m/z 283.2 and their product ions at m/z 233.2 were used as monitor ions for detecting JHSB3 and 10S-JHSB3. The lowest detection limit of JHSB3 in our methodology is 0.25 pg [19]. Ten µL of the CA product in methanol was used.

The figures show charts of chiral ultra-performance liquid chromatography coupled with tandem mass spectrometry of the corpus allatum products from various heteropteran species. The vertical axis indicates the signal intensity of the product ion at m/z 233.2 produced from the precursor ion at m/z 283.2.

保幼激素（Juvenile hormone, JH）在昆虫发育与繁殖的几乎所有环节中均发挥关键调控作用。保幼激素属于一类无环倍半萜类化合物，其法尼醇骨架经化学修饰后，在部分昆虫演化支系中形成了一系列同源激素。保幼激素III（环氧法尼酸甲酯，epoxidised methyl farnesoate）是昆虫中分布最广泛的保幼激素，但鳞翅目（蝶与蛾类）和‘高等’双翅目（短角亚目：Brachycera；蝇类）则演化出了各自独有的保幼激素。尽管保幼激素最初在半翅目异翅亚目（true bugs）中被提出，但异翅类保幼激素的化学结构直至近年才得以解析。此外，近期研究在部分异翅类昆虫中发现了一种新型保幼激素——保幼激素III双环氧跳过型（JH III skipped bisepoxide, JHSB3），但其分类学分布范围仍未明确。本研究通过超高效液相色谱-串联质谱（ultra-performance liquid chromatography coupled with tandem mass spectrometry, UPLC-MS/MS）技术，对覆盖几乎所有异翅类演化支系的31个异翅目物种的JHSB3合成情况开展了检测。结果显示，所有受试物种均能合成JHSB3，表明该激素在异翅类昆虫中广泛存在，且其演化起源可追溯至异翅亚目的共同祖先。

方法
如需进一步阐释与参考文献，请参见本文正文（Matsumoto等，2021，《皇家学会开放科学》（Royal Society Open Science））。

昆虫样本采集自日本大阪府、奈良县、京都府与冈山县的野外，或从本实验室及合作实验室的饲养种群中获取（详见附表S1与附图S1）。本研究仅使用成虫个体。由于野外采集的个体数量有限，其中14个物种的实验结果基于单头样本。将昆虫在冰上麻醉后，用黏土固定。参照既往研究[25]中的方法，摘除附着有心侧体（corpora cardiaca）的咽侧体（corpus allatum, CA）。简言之，将咽侧体置于30~50 μL改良型最小必需培养基（Minimum Essential Medium）中，于30 ℃下孵育5小时。随后用正己烷萃取保幼激素，经氩气流吹干后，重新溶解于30~1200 μL甲醇中（详见附表S1）。

本研究使用配备手性色谱柱（CHIRALPAK IA-U，3.0 mm × 100 mm，粒径1.6 μm，大赛璐，日本东京）的超高效液相色谱-串联质谱系统（ACQUITY UPLC H-Class, Xevo TQ-S micro，沃特世，美国马萨诸塞州米尔福德），通过比对JHSB3的保留时间完成定性分析[18,19]。参照既往方法合成了标准品JHSB3与10S-JHSB3[15]。对标准品JHSB3的质谱分析显示，其准分子离子峰[M+H]+的质荷比为283.2，加钠峰[M+Na]+为305.3；以质荷比283.2的离子为前体离子时，可检测到质荷比42.9与233.2的产物离子，而以质荷比305.3的离子为前体时则无碎裂峰产生。本研究以质荷比283.2的离子及其产物离子m/z 233.2作为监测离子，用于检测JHSB3与10S-JHSB3。本方法对JHSB3的最低检测限为0.25 pg[19]。实验时取10 μL溶解于甲醇中的咽侧体孵育产物进样。

本研究附图为不同异翅类物种咽侧体孵育产物的手性超高效液相色谱-串联质谱图。纵坐标代表以质荷比283.2的前体离子生成的产物离子m/z 233.2的信号强度。