Autonomous regulation of the insect gut by circadian genes acting downstream of juvenile hormone signaling

Abstract

In temperate regions, the shortening day length informs many insect species to prepare for winter by inducing diapause. The adult diapause of the linden bug, Pyrrhocoris apterus, involves a reproductive arrest accompanied by energy storage, reduction of metabolic needs, and preparation to withstand low temperatures. By contrast, nondiapause animals direct nutrient energy to muscle activity and reproduction. The photoperiod-dependent switch from diapause to reproduction is systemically transmitted throughout the organism by juvenile hormone (JH). Here, we show that, at the organ-autonomous level of the insect gut, the decision between reproduction and diapause relies on an interaction between JH signaling and circadian clock genes acting independently of the daily cycle. The JH receptor Methoprene-tolerant and the circadian proteins Clock and Cycle are all required in the gut to activate the Par domain protein 1 gene during reproduction and to simultaneously suppress a mammalian-type cryptochrome 2 gene that promotes the diapause program. A nonperiodic, organ-autonomous feedback between Par domain protein 1 and Cryptochrome 2 then orchestrates expression of downstream genes that mark the diapause vs. reproductive states of the gut. These results show that hormonal signaling through Methoprene-tolerant and circadian proteins controls gut-specific gene activity that is independent of circadian oscillations but differs between reproductive and diapausing animals.

Fig. 1.

The cry2 and Pdp1_iso1 genes are inversely regulated under diapause and reproductive conditions in the gut of P. apterus females. (A) Reproductive diapause in P. apterus females. Ovaries are small and contain no maturing oocytes under SD condition (Left) in which females naturally lack JH. Oogenesis commences upon extending the photoperiod (i.e., LD; Right) or after JH mimic treatment. (B) Levels of cry2 mRNA in the gut remain high under diapause and low during the reproductive phase (Left) irrespective of daily fluctuations (Right). (C) The Cry2 protein is detected in cell nuclei of the gut epithelium of diapause females (Left) but not in reproductive females. (D) CDL experiment (Methods) shows that cry2 expression in the gut depends on the reproductive state rather than on the photoperiod. Females that remained nonreproductive (i.e., diapause) after 2 wk of exposure to CDL (16.5 h light, 7.5 h dark) expressed high cry2 levels, whereas those that became reproductive under the same CDL conditions showed down-regulation of cry2. (E) Expression of Pdp1_iso1 follows a pattern opposite to that of cry2. (F) Expression of cry2 and Pdp1_iso1 transcripts switched to the diapause mode between 5 and 7 d after transfer from LD to SD photoperiod. (G) Immunoblot shows that expression of Cry2 protein in the gut of females experiencing diapause was depleted by cry2 RNAi. Low Cry2 levels occurring in reproductive females increased upon Met RNAi. Levels of mRNAs in B, D, E, and F were determined by using qRT-PCR and were normalized to rp49 expression; data are mean ± SEM from three independent experiments (error bars omitted in F for clarity).

Fig. 2.

Factors required for the reciprocal cry2 and Pdp1_iso1 regulation in the gut. (A) Reproductive females that naturally produce JH were injected with dsRNA to silence the indicated genes, and the levels of cry2 and Pdp1_iso1 mRNAs in their guts were determined 4 d later, together with ovarian morphology. lacZ dsRNA served as a control. Depletion of Met, Clk, and Cyc (individually or in combinations) altered both transcripts toward their diapause mode; depletion of Met or Tai prevented oogenesis. (B) RNAi depletion of Met, Clk, or Cyc in diapause females partially reduced cry2 and increased Pdp1_iso1 mRNAs, equalizing their levels. (C) Met, Clk, and Cyc are necessary for the JH mimic methoprene to revert the levels of cry2 and Pdp1_iso1 mRNAs to the reproductive mode, as both transcripts became approximately equalized in the guts of Met, Clk, or cyc RNAi females that were given methoprene. (D and E) Pdp1_iso1 and cry2 form a feedback loop of mutual repressors in the gut. dsRNAs were injected to females 1 d after adult ecdysis, and transcript levels in their guts were monitored 1, 2, 3, 5, 7, 14, and 21 d later. Knockdown of either gene remained effective throughout this period. Removal of Cry2 in diapause females caused Pdp1_iso1 mRNA to increase near levels normally occurring in guts of reproductive females (D). Conversely, expression of cry2 was enhanced when reproductive females were subjected to Pdp1_iso1, although to a lesser extent than in diapause controls. Values are mean ± SEM from three independent experiments. (*P < 0.05 and **P < 0.001 vs. lacZ controls as assessed, Tukey honestly significant difference test).

Fig. 3.

Regulation of genes downstream of Cry2, Pdp1_iso1, and JH/Met in the gut. (A) Reproductive (i.e., LD) and diapause (i.e., SD) females were injected with dsRNAs and, after 4 d, treated with methoprene as indicated below the columns. Four days later, mRNA levels were measured in their guts for Pdp1_iso1, lip, def, and est genes, whose expression characterizes the reproductive state (Upper), and for cry2, tf, and sod genes, which are active under diapause (Lower). (B) Expression of the reproduction and diapause downstream genes in isolated guts. Guts were dissected from control or from diapause (i.e., SD) females 2 d after dsRNA injection, and were cultured for 2 d with or without methoprene as indicated. Alternatively, isolated guts were exposed to cry2 dsRNA for 2 d in culture (asterisk). All data have been normalized to rp49 expression and are shown relative to the levels of reproduction downstream genes in untreated LD females (Upper) and to the levels of diapause downstream genes in untreated SD females (Lower), respectively, that were set to 100%. Statistical significance of the differences is shown in Table S1.

Fig. 4.

Regulation of the reproductive/diapause status of the insect gut. Under LD conditions, which favor reproduction (Left), the corpora allata (CA) secrete JH in response to signals from the pars intercerebralis (PI) of the brain. In the gut, JH acts through its receptor Met, Clk, and Cyc to stimulate expression of Pdp1_iso1 and other genes that characterize the reproductive state, whereas expression of cry2 and diapause downstream genes is suppressed. Although the regulation involves circadian clock genes, it is independent of daily oscillations of the circadian gene expression. In the absence of endogenous JH under short photoperiod (Right), expression of cry2 prevails over Pdp1_iso1, favoring the diapause-specific program. By acting through Met, Clk, and Cyc, exogenous JH mimic can induce the reproductive program under SD condition.