Introduction
Juvenile hormone (JH), synthesized at and released from the corpus allatum (CA), an endocrine gland, plays a key role in almost every aspect of insect development and reproduction (Nijhout, Reference Nijhout1994; Goodman and Cusson, Reference Goodmann, Cusson and Gilbert2012; Jindra et al., Reference Jindra, Palli and Riddiford2013, Reference Jindra, Bellés and Shinoda2015; Roy et al., Reference Roy, Saha, Zou and Raikhel2018; Santos et al., Reference Santos, Humann and Harftfelder2019). The JH structure was first determined from the cecropia moth Hyalophora cecropia, by Röller et al. (Reference Röller, Dahm, Sweeley and Trost1967). The cecropia JH is now referred to as JH I, as thereafter, various JHs such as JH 0, JH II, JH III, and JHB3 were identified from various insect orders (Jindra et al., Reference Jindra, Palli and Riddiford2013).
The Heteroptera (true bugs), a suborder of Hemiptera, represent over 45,000 described species and are part of the successful radiation of hemimetabolous insects (Weirauch and Schuh, Reference Weirauch and Schuh2011; Foottit and Adler, Reference Foottit and Adler2017). Although the JH of heteropteran species has long been controversial (Bowers et al., Reference Bowers, Marsella and Evans1983; Baker et al., Reference Baker, Tsai, Reuter and Schooley1988; Numata et al., Reference Numata, Numata, Takahashi, Nakagawa, Iwatani, Takahashi, Miura and Chinzei1992; Kotaki, Reference Kotaki1993; Reference Kotaki1996; Miyawaki et al., Reference Miyawaki, Tanaka and Numata2006; Teal et al., Reference Teal, Jones, Jones, Torto, Nyasembe, Borgemeister, Alborn, Kaplan, Boucias and Lietze2014), Kotaki et al. (Reference Kotaki, Shinada, Kaihara, Ohfune and Numata2009, Reference Kotaki, Shinada, Kaihara, Ohfune and Numata2011) identified methyl (2R,3S,10R)-2,3;10,11-bisepoxyfarnesoate as a novel JH in Plautia stali with a chiral GC-MS analysis and named it juvenile hormone III skipped bisepoxide (JHSB3). After those landmark studies, the presence of JHSB3 was investigated in several heteropteran species, i.e., Pyrrhocoris apterus (Hejnikova et al., Reference Hejnikova, Paroulek and Hodkova2016), Dipetalogaster maxima and Oncopeltus fasciatus (Ramirez et al., Reference Ramirez, Nouzova, Michalkova, Fernandez-Lima and Noriega2020), Riptortus pedestris (Lee et al., Reference Lee, Kim, Jang, Kim, Kotaki, Shinoda, Shinada, Yoo and Lee2019), and Rhodnius prolixus (Villalobos-Sambucaro et al., Reference Villalobos-Sambucaro, Nouzova, Ramirez, Alzugaray, Fernandez-Lima, Ronderos and Noriega2020). The analysis was performed by high-performance liquid chromatography-mass mass analysis (HPLC-MS/MS) with a C18 reverse-phase column. The presence of JHSB3 was proposed by the identification of the retention times of natural and authentic JHSB3. However, there are three stereoisomers, (2R,3S,10S), (2S,3R,10R), and (2S,3R,10S) for JHSB3, and these stereoisomers could not be separated on the C18 stationary phase (Ando et al., Reference Ando, Matsumoto, Misaki, Mano, Shiga, Numata, Kotaki, Shinada and Goto2020). In addition, Kotaki et al. reported the presence of not only JHSB3 but also 10S-JHSB3 in the CA product of Halyomorpha halys. The latter is a new JH in insects (Kotaki et al., Reference Kotaki, Matsumoto, Kaihara, Ando, Misaki and Shinada2020). These results indicated that analytical conditions to discriminate JHSB3 and its 10S-isomer are needed to determine the JH of heteropteran species.
The cabbage bug Eurydema rugosa Motschulsky (Pentatomidae) is widely distributed in East Asia and known as a pest of wild and cultivated crucifers, such as Chinese cabbage, Japanese radish, and wasabi (Tomokuni et al., Reference Tomokuni, Yasunaga, Takai, Yamashita, Kawamura and Kawasawa1993; Schaefer and Panizzi, Reference Schaefer and Panizzi2000). This species has two facultative adult diapauses, and their environmental cues are photoperiod and food type (leaves or seeds). One of the diapauses is induced by feeding on seeds under long-day conditions (summer diapause), and the other is induced by short-day conditions irrespective of the food type (leaves or seeds) (winter diapause) (Numata and Yamamoto, Reference Numata and Yamamoto1990). It is generally accepted in various insect species that a lack of JH triggers adult diapause and JH biosynthesis promotes its termination (Denlinger et al., Reference Denlinger, Yocum, Rinehart and Gilbert2012). In heteropterans, the significance of JH in diapause regulation was revealed by allatectomy and silencing of the genes involved in the JH-signalling cascade by RNA interference (Kotaki and Yagi, Reference Kotaki and Yagi1989; Morita and Numata, Reference Morita and Numata1997; Bajgar et al., Reference Bajgar, Jindra and Dolezel2013; Smykal et al., Reference Smykal, Bajgar, Provaznik, Fexova, Buricova, Takaki, Hodkova, Jindra and Dolezel2014). The topical application of JH is also widely used to see its effect. However, JH analogues (JHA) or non-innate JHs have been used in such experiments with heteropterans (Kotaki and Yagi, Reference Kotaki and Yagi1989; Adams et al., Reference Adams, Filipi and Yi2002; Miyawaki et al., Reference Miyawaki, Tanaka and Numata2006; Cho et al., Reference Cho, Lee, Kim and Boo2007; Ikeno et al., Reference Ikeno, Tanaka, Numata and Goto2010; Bajgar et al., Reference Bajgar, Jindra and Dolezel2013; Smykal et al., Reference Smykal, Bajgar, Provaznik, Fexova, Buricova, Takaki, Hodkova, Jindra and Dolezel2014; Urbanová et al., Reference Urbanová, Bazalová, Vanĕčková and Dolezel2016; Penca and Hodges, Reference Penca and Hodges2017) simply because the heteropteran JH has long been physiochemically unidentified. Experiments with JHA advanced our knowledge on JH roles, but methoprene, one of the most well-known JHAs, is not effective in pentatomid bugs (Kotaki, Reference Kotaki1996). Thus, we have to be careful about interpreting the JHA results. Experiments with the innate JH, JHSB3, are still very limited (Kotaki et al., Reference Kotaki, Shinada, Kaihara, Ohfune and Numata2011, Reference Kotaki, Matsumoto, Kaihara, Ando, Misaki and Shinada2020; Ando et al., Reference Ando, Matsumoto, Misaki, Mano, Shiga, Numata, Kotaki, Shinada and Goto2020).
In the present study, we investigated the chemical structure of the JH in E. rugosa, to expand our knowledge of the heteropteran JH and its role in diapause regulation. From the culture media of the CA attached with the corpora cardiaca (CC) of E. rugosa, the JH was physicochemically analysed by a chiral ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) that we introduced very recently to JH research (Ando et al., Reference Ando, Matsumoto, Misaki, Mano, Shiga, Numata, Kotaki, Shinada and Goto2020; Kotaki et al., Reference Kotaki, Matsumoto, Kaihara, Ando, Misaki and Shinada2020; Matsumoto et al., Reference Matsumoto, Yasuno, Yasuda, Hayashi, Goto and Shinada2020). Further, its juvenilizing activity was determined in last instar nymphs, and its effects on the termination of both the summer and winter diapauses were investigated.
Materials and methods
Insects
Adults of E. rugosa were collected from brown mustard Brassica juncea on the banks of the Yamatogawa River (34.59°N, 135.51°E), from March to April in 2018. The insects were reared in groups under long-day conditions (LD 16:8 h) at 25 ± 1 °C on seeds of rape Sinapis alba, with tap water. Their offspring were reared in the same manner and used in the experiments unless otherwise stated.
JH identification by UPLC-MS/MS
Fourteen of the E. rugosa males that were collected were utilized for the CC-CA extraction and incubation, performed according to the methods described in Matsumoto et al. (Reference Matsumoto, Numata and Shiga2013). In brief, the insects were anaesthetized on ice and immobilized with clay and were dissected in 0.9% NaCl. All the CC-CA complexes were incubated in 40 μl of minimum essential medium (with Hank's salt and L-glutamate and without sodium bicarbonate) added with 20 mM of HEPES and 5 ppm of Tween 80 and adjusted to pH 7.2 at 30 °C for 5 h in a glass tube (6 mm in diameter, 30 mm in height). After adding 60 μl of hexane, the supernatant was extracted. This hexane extraction was repeated three times and then the solvent was replaced with methanol.
The UPLC-MS/MS (ACQUITY UPLC H-Class, Xevo TQ-S micro, Waters, Milford, MA) and a chiral column (CHIRALPAK IA-U, 100 mm in length, Daicel, Tokyo, Japan) were used to compare the retention times of JHSB3, its stereoisomers, JH I, JH III, and the CC-CA extract. The system operation, data acquisition, and analysis were controlled and processed by MassLynx software according to Ando et al. (Reference Ando, Matsumoto, Misaki, Mano, Shiga, Numata, Kotaki, Shinada and Goto2020). In brief, the flow rate of each sample through the chiral column was 0.4 ml min−1 in the solvent (15% water and 85% methanol). The column temperature was 30 °C. The mass spectrometer was operated in the positive ion mode. The tuning parameters were optimized for JHSB3 and its stereoisomers: desolvation temperature 400 °C, desolvation gas flow 800 l h−1, cone voltage 20 V, collision energy 10 V. For JH III, collision energy of 25 V was used. Authentic JHSB3, its stereoisomers, and JH III were synthesized as described in Kotaki et al. (Reference Kotaki, Shinada, Kaihara, Ohfune and Numata2009) and JH I was synthesized as described in Manabe et al. (Reference Manabe, Ohfune and Shinada2012). 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 its product ions at m/z 233.2 were used as monitor ions for detecting JHSB3 and its stereoisomers. For JH I and JH III, ions at m/z 294.5 and 261.3 and its product ions at m/z 263.3 and 43.0 were used as monitor ions, respectively.
The juvenilizing activity of JHSB3
Nymphs were reared from the egg stage under short-day conditions (LD 10:14 h) at 25 ± 1 °C in a group of 30 individuals in a plastic cup (75 mm in diameter, 40 mm in depth). Nymphs that had ecdysed to the last (5th) instar stage were collected within 48 h from the ecdysis and 0.6 μl of hexane (solvent) or various concentrations of JHSB3 in hexane were applied to the dorsal side of the abdomen using a 10 μl-syringe. After the application, five or fewer individuals per group were reared in individual Petri dishes (85 mm in diameter, 15 mm in depth) and maintained under the same photoperiodic and temperature conditions. After the final moult, scutellum length, forewing length, and pronotum width were measured using Nikon NIS Elements BR 3.0 software (Nikon, Tokyo, Japan) and the relative lengths of the scutellum and forewing to the pronotum width were calculated to evaluate the juvenilizing effects according to Kotaki (Reference Kotaki1996) and Kotaki et al. (Reference Kotaki, Shinada, Kaihara, Ohfune and Numata2011). The juvenilizing effect was also assessed from the colour patterns on the dorsal abdomen. Individuals with black semi-ellipses on the yellowish background were regarded as the nymphal type, whereas individuals with yellowish spots on the black background were regarded as the adult type.
The diapause-terminating activity of JHSB3
To induce summer and winter diapauses, the insects were reared under long-day conditions (LD 16:8 h) and short-day conditions (LD 10:14 h), respectively, at 25 ± 1 °C from the egg stage with rape seeds. Nymphs were reared in a group of 50 individuals in a plastic pot (145 mm in diameter, 90 mm in depth). After the final moult, adult females were reared in groups of 5 or fewer individuals per Petri dish. Thirty days after the final moult, we topically applied 2 μl of hexane (solvent) or various concentrations of JHSB3 in hexane (the first application) on the females in both summer and winter diapauses and returned them to their respective conditions. Five days later, the same amount of hexane or JHSB3 was applied (the second application). The diapause-terminating activity was assessed 40 days after the final moult by assessing ovarian status. Females with a yolk deposition in the oocytes were judged to be reproductive and those with no deposition were judged to be non-reproductive (diapause) according to Numata and Yamamoto (Reference Numata and Yamamoto1990).
Statistical analysis
Treatment was treated as the independent variable. Relative scutellum and forewing lengths were treated as dependent variables. They were not normally distributed and were analysed with the Steel-Dwass test (Zar, Reference Zar2010) to perform nonparametric multiple comparisons. The proportions were treated as dependent variables and were analysed with Tukey-type multiple comparisons for proportions (Zar, Reference Zar2010).
Results
UPLC-MS/MS analysis of the CA product
UPLC-MS/MS data of JHSB3, its stereoisomers, and the CA product of E. rugosa are depicted in fig. 1. The retention times of the main peak of the CA product (3.49 min), as well as that of co-injection of the CA product and JHSB3, were identical with that of JHSB3. On the other hand, the retention times of the stereoisomers were different from that of the CA product (fig. 1). In addition, peaks corresponding to JH I and JH III were undetectable in the CA product (data not shown). These results indicate that the CA of E. rugosa produces and secretes JHSB3.
Figure 1. Chiral ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) analyses of the corpus allatum (CA) product of Eurydema rugosa, juvenile hormone III skipped bisepoxide (JHSB₃), their mixture, and stereoisomers of JHSB3. The vertical axis indicates the signal intensity for m/z 233.2 [M + H]+. Chemical structures of JHSB₃ and its stereoisomers are also shown.
Juvenilizing effect of JHSB3 on last instar nymphs
Because we detected only JHSB3 in the CA product of E. rugosa, we topically applied JHSB3 to the last instar nymphs to investigate its biological activity. After the final moult, scutellum length, forewing length, and pronotum width were measured and the relative lengths of the scutellum and forewing to the pronotum width were calculated (fig. 2A). Topical application of JHSB3 inhibited metamorphosis in a dose-dependent manner. Relative lengths of the forewing and scutellum were approximately 0.60 and 0.39, respectively, in the last instar nymphs and these values were twice as small as the relative lengths in the adult females (1.37 and 0.69, respectively) (fig. 2B). Topical application of hexane (solvent) and a very small amount of JHSB3 showed no juvenilizing effect. However, 0.06 μg of JHSB3 significantly lowered the values (Steel-Dwass test, P < 0.05). These values were further lowered by 0.6 and 1.0 μg of JHSB3 (Steel-Dwass test, P < 0.05).
Figure 2. Juvenilizing effects of juvenile hormone III skipped bisepoxide (JHSB₃) in Eurydema rugosa. (A) Parameters for the juvenilizing activity of JHSB3. A last instar nymph (a), an adult (b), an adult of which wings were removed (c), and a nymph-adult intermediate (d), obtained as a result of the application of a JH-active sample. Pronotum, forewing, and scutellum lengths indicated in a and b were measured. The colour patterns of the dorsal abdomen, shown in the framed box in a and c, were also observed. Scale bar indicates 3 mm. (B) Juvenilizing effects of JHSB₃ on the relative forewing (upper panel) and scutellum (lower panel) lengths in females. The solvent application to the nymph was also shown as a control. Each datum point and error bar indicate median ± interquartile range. The same letters indicate no statistical differences among the samples (Steel-Dwass test, P > 0.05). (C) Juvenilizing effect of JHSB₃ on the colour patterns of the dorsal abdomen in females. Their colour patterns were categorized into 2 types; adult and nymphal types. The solvent application to the nymph was also shown as a control. The same letters indicate no statistical differences among the samples (Tukey-type multiple comparisons for proportions, P > 0.05).
The colour patterns of the dorsal abdomen in adult females and nymphs were largely different (fig. 2A). As the amount of JHSB3 applied increased, the colour patterns became closer to the nymphal type (fig. 2C; Tukey-type multiple comparisons for proportions, P < 0.05).
Effect of JHSB3 on ovarian development in females in summer and winter diapauses
None of the females developed their ovaries when the solvent or 0.002 μg of JHSB3 were applied, irrespective of summer and winter diapauses (fig. 3). Small proportions of individuals developed their ovaries when they were treated with 0.02 μg of JHSB3. The proportions were not statistically different between summer and winter diapauses (Tukey-type multiple comparisons for proportions, P > 0.05). When higher doses of JHSB3 were applied, most females developed their ovaries, irrespective of the diapause type (Tukey-type multiple comparisons for proportions, P < 0.05). These results indicate that JHSB3 effectively terminates both summer and winter diapauses.
Figure 3. Effects of juvenile hormone III skipped bisepoxide (JHSB3) on ovarian development in Eurydema rugosa females in summer (S) and winter (W) diapauses. The solvent application to the nymph was also shown as a control. The same letters indicate no statistical differences among the samples (Tukey-type multiple comparisons for proportions, P > 0.05).
Discussion
Our chemical analysis using the authentic JHSB3 and their diastereoisomers by chiral UPLC-MS/MS clarified that E. rugosa synthesizes and secretes JHSB3, but not its stereoisomers. Neither JH III nor JH I was detected in the CA product. The topical application of the synthetic JHSB3 to the last instar nymphs inhibited their metamorphosis and induced nymphal-type colouration of the dorsal abdomen, in a dose-dependent manner. In addition, topical application of JHSB3 effectively terminated summer and winter diapauses in females. It is important to note that the effective doses were comparable to those of the previous study, that determined the role of JHSB3 in P. stali and R. pedestris (Kotaki et al., Reference Kotaki, Shinada, Kaihara, Ohfune and Numata2011; Ando et al., Reference Ando, Matsumoto, Misaki, Mano, Shiga, Numata, Kotaki, Shinada and Goto2020). These data suggest that JHSB3 is the JH of E. rugosa. Further investigations of its presence and the fluctuations of its haemolymph concentrations are required in the future study.
The present study revealed that the topical application of JHSB3 effectively terminated both summer and winter diapauses. The topical application of JHA (Cho et al., Reference Cho, Lee, Kim and Boo2007; Bajgar et al., Reference Bajgar, Jindra and Dolezel2013; Smykal et al., Reference Smykal, Bajgar, Provaznik, Fexova, Buricova, Takaki, Hodkova, Jindra and Dolezel2014; Urvanová et al., Reference Urbanová, Bazalová, Vanĕčková and Dolezel2016; Penca and Hodges, Reference Penca and Hodges2017) and that of JHSB3 (Kotaki et al., Reference Kotaki, Shinada, Kaihara, Ohfune and Numata2011; Ando et al., Reference Ando, Matsumoto, Misaki, Mano, Shiga, Numata, Kotaki, Shinada and Goto2020) also terminated adult diapause of several heteropterans. Although we have not investigated its haemolymph concentration in E. rugosa females in diapause, the results are consistent with the general concept that the lack of JH triggers adult diapause and the resumption of JH biosynthesis promotes diapause termination (Denlinger et al., Reference Denlinger, Yocum, Rinehart and Gilbert2012). Individuals in summer and winter diapauses are physiologically distinct in E. rugosa; winter diapause can be terminated by a low-temperature exposure, whereas the exposure is insufficient for terminating summer diapause (Ikeda-Kikue and Numata, Reference Ikeda-Kikue and Numata1994). The nearly identical doses of JHSB3 required for terminating both summer and winter diapauses in the present study suggest that the differences in the responsiveness to the environmental factors are based, not on the responsiveness to the JH occurring during the diapause-termination process, but on the process governing activation of the CA or its upstream cascades.
As with females, E. rugosa males also have two facultative adult diapauses (summer and winter diapauses). Numata and Yamamoto (Reference Numata and Yamamoto1990) revealed that the testis is largest in directly-developed adult males (non-diapause), smallest in males in summer diapause, and intermediate in males in winter diapause. The general cause for diapause, the lack of JH, cannot explain the size difference between diapauses. Unknown hormonal factors in addition to JH may be involved in these diapauses in E. rugosa. It is of interest to identify these factors and investigate the additive or synergistic effects of JHSB3 and the factors on diapauses in future studies.
Acknowledgements
We thank Drs Kayoko Ikeda-Kikue and Hideharu Numata for their invaluable advice during this study. We thank Editage (www.editage.jp) for the English language editing of the manuscript submitted for publication.
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