Photoperiod and temperature separately regulate nymphal development through JH and insulin/TOR signaling pathways in an insect

Abstract

Insects living in the temperate zone enter a physiological state of arrested or slowed development to overcome an adverse season, such as winter. Developmental arrest, called diapause, occurs at a species-specific developmental stage, and embryonic and pupal diapauses have been extensively studied in mostly holometabolous insects. Some other insects overwinter in the nymphal stage with slow growth for which the mechanism is poorly understood. Here, we show that this nymphal period of slow growth is regulated by temperature and photoperiod through separate pathways in the cricket Modicogryllus siamensis The former regulates the growth rate, at least in part, through the insulin / target of rapamycin (TOR) signaling pathway. Lower temperature down-regulates the expression of insulin-like peptide (Ms'Ilp) and Target of rapamycin (Ms'Tor) genes to slow down the growth rate without affecting the number of molts. The latter regulates the number of molts independent of temperature. Short days increase the number of molts through activation of the juvenile hormone (JH) pathway and down-regulation of myoglianin (Ms'myo), a member of the TGFβ family, which induces adult metamorphosis. In contrast, long days regulate Ms'myo expression to increase during the fifth to sixth instar to initiate adult metamorphosis. When Ms'myo expression is suppressed, juvenile hormone O-methyl transferase (Ms'jhamt) was up-regulated and increased molts to prolong the nymphal period even under long-day conditions. The present findings suggested that the photoperiod regulated Ms'myo, and the JH signaling pathway and the temperature-controlled insulin/TOR pathway cooperated to regulate nymphal development for overwintering to achieve seasonal adaptation of the life cycle in M. siamensis.

Keywords: insulin/TOR signaling pathway; myo; nymphal development; photoperiod; temperature.

Fig. 1.

Effects of photoperiod and temperature on nymphal development in the cricket Modicogryllus siamensis under long-day (LD, 16L:8D) or short-day conditions (SD, 12L:12D) at 25 °C and 30 °C. (A and B) Adult emergence patterns after hatching from eggs (A) and proportions of molt numbers before adult emergence (B). The number of animals used was 84, 46, 92, and 29 for LD 30 °C, LD 25 °C, SD 30 °C, and SD 25 °C, respectively. Numbers in different color fractions in B indicate the number of molts. (C) Adult body weight (mean ± SEM) measured at 1 d after adult emergence. Values with different letters significantly differ from each other (Tukey test, P < 0.05). Numbers in parenthesis indicate the number of animals used. (D) Photographs of LD and SD adult crickets raised at 30 °C. (Scale bar, 1 cm.)

Fig. 2.

Involvement of Ms’myo in photoperiodic regulation of nymphal development in the cricket Modicogryllus siamensis. (A and B) Expression patterns of Ms’myo (A) and Ms’jhamt (B) mRNAs during nymphal development under long-day (LD, 16L:8D, circles) and short-day (SD, 12L:12D, squares) conditions at 25 °C (blue) or 30 °C (orange). Ms’myo mRNA levels were significantly higher under LD than SD for the last three instar nymphs. Ms’jhamt levels tended to be higher under SD throughout the tested period. Values were an average of four samples, including three heads each, and error bars indicate SEM. a and b indicate a significant difference between LD and SD at 25 °C and 30 °C, respectively. (C–F) Effects of Ms’myo nymphal RNAi (Ms’myo^nRNAi) on adult emergence under LD (C) and SD (D), molt numbers (E), and body weights (F) at 25 °C. Gray and green symbols in C, D, and F indicate DsRed2^nRNAi control and Ms’myo^nRNAi crickets, respectively. N and numbers in parenthesis indicate the number of animals used. Numbers in E indicate the number of molts. Ms’myo^nRNAi significantly delayed adult emergence (U test, P < 0.001) and increased molt numbers compared with that of DsRed2^nRNAi crickets (U test, P < 0.001). Body weights were significantly increased in Ms’myo^nRNAi-treated crickets under LD (Tukey test, P < 0.05). Values with different letters significantly differ from each other.

Fig. 3.

Effects of dsMs’myo (A), methoprene treatments (B), or NCC I+II severance (C) on the relative abundance of Ms’myo, Ms’jhamt, and Ms’Kr-h1 mRNAs in the head of Modicogryllus siamensis. Values are an average of four samples containing three heads each, shown relative to those of the DsRed2^RNAi controls (A and B) or those of sham operated crickets (C). Males and females were pooled. The abundance of Ms’rp49 mRNA was used as an internal reference. Error bars indicate SEM. Asterisks represent significant differences between control and treated crickets: **P < 0.01, *P < 0.05, t test. (A) mRNA levels were measured on day 1 in the sixth instar nymphs treated with dsDsRed2 (black) or dsMs’myo (light green) on day 1 in the fourth instar under LD (16L:8D) at 30 °C. The samples were collected at 6 h after light-on (ZT6). Ms’myo^nRNAi significantly suppressed Ms’myo mRNA levels, whereas Ms’jhamt and Ms’Kr-h1 levels were significantly up-regulated. (B) mRNA levels were measured on day 3 in the sixth instar nymphs, which were treated with methoprene on day 3 of the fifth instar. Methoprene up-regulates Ms’jhamt and Ms’Kr-h1 expression but had no clear effect on Ms’myo levels. (C) mRNA levels were measured 24 h after the NCC I+II severance (NCCX) on day 1 of the seventh instar nymphs. The operation resulted in the up-regulation of Ms’myo and down-regulation of Ms’jhamt and Ms’Kr-h1. For further explanations, see text.

Fig. 4.

Involvement of the insulin/TOR signaling pathway in growth rate regulation by temperature in Modicogryllus siamensis. (A) Expression patterns of Ms’Ilp (a), Ms’Inr (b), and Ms’Tor (c) mRNA levels during nymphal development under long-day (LD, 16L:8D, circles) and short-day (SD, 12L:12D, squares) conditions at 25 °C (blue) and 30 °C (orange). Different letters indicate that values are significantly different from each other at the same stage (Tukey test, P < 0.05). Colored letters indicate the category of the value labeled with the same color symbol. Black letters indicate that values closely located are in the same category. (B) Suppression of the growth rate by Ms’Inr^nRNAi under LD and SD at 30 °C. Under both LD and SD, Ms’Inr^nRNAi-treated crickets (brown column) showed less of an increase in body weight compared with that of DsRed2^nRNAi-treated controls (gray column). **P < 0.01, t test. Error bars in A and B indicate SEM. (C) Adult emergence was delayed by Ms’Inr^nRNAi treatment. Brown and gray symbols indicate Ms’Inr^nRNAi-treated and DsRed2^nRNAi-treated crickets, respectively. Circles and squares indicate results under LD and SD, respectively. (D) Molt numbers of Ms’Inr^nRNAi-treated and DsRed2^nRNAi-treated crickets. Under LD, Ms’Inr^nRNAi significantly increased the ratio of crickets with 8 molts (U test, P < 0.01) but never induced a ninth or later molt. No significant changes were induced under SD (U test, P > 0.18).

Fig. 5.

A scheme of the photoperiodic control of nymphal development in the cricket Modicogryllus siamensis. NSC; neurosecretory cells. Dark red and blue lines indicate long-day (LD) and short-day (SD) pathways, respectively. LDs stimulate Ms’myo expression during the fifth to seventh instar stage, which suppresses Ms’jhamt expression and reduces JH synthesis, leading to adult emergence. SDs suppress Ms’myo expression and stimulate Ms’jhamt expression during the same stage, which increases JH production, resulting in nymphal molts, hence leading to slow nymphal development. Temperature regulates the growth rate through the insulin/TOR signaling pathway to accelerate or slow down at higher or lower temperatures, respectively. Slow development for overwintering is most likely maintained by short photoperiods and cold temperatures during autumn to winter. For further explanations, see text.