Methoprene-tolerant and Krüppel homolog 1 are actors of juvenile hormone-signaling controlling the development of male sexual behavior in the moth Agrotis ipsilon.

In insects, juvenile hormone (JH) is critical for the orchestration of male reproductive maturation. For instance, in the male moth, Agrotis ipsilon, the behavioral response and the neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs), to the female-emitted sex pheromone increase with fertility during adulthood and the coordination between these events is governed by JH. However, the molecular basis of JH action in the development of sexual behavior remains largely unknown. Here, we show that the expression of the paralogous JH receptors, Methoprene-tolerant 1 and 2 (Met1, Met2) and of the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) within ALs raised from the third day of adult life and this dynamic is correlated with increased behavioral responsiveness to sex pheromone. Met1-, Met2- and Kr-h1-depleted sexually mature males exhibited altered sex pheromone-guided orientation flight. Moreover, injection of JH-II into young males enhanced the behavioral response to sex pheromone with increased AL Met1, Met2 and Kr-h1 mRNA levels. By contrast, JH deficiency suppressed the behavioral response to sex pheromone coupled with reduced AL Met1, Met2 and Kr-h1 mRNA levels in allatectomized old males and these inhibitions were compensated by an injection of JH-II in operated males. Our results demonstrated that JH acts through Met-Kr-h1 signaling pathway operating in ALs, to promote the pheromone information processing and consequently the display of sexual behavior in synchronization with fertility to optimize male reproductive fitness. Thus, this study provides insights into the molecular mechanisms underlying the hormonal regulation of reproductive behavior in insects.

Involvement of Methoprene-tolerant and Krüppel homolog 1 in juvenile hormone-signaling regulating the maturation of male accessory glands in the moth Agrotis ipsilon.

Male accessory glands (MAGs) produce seminal fluid proteins that are essential for the fertility and also influence the reproductive physiology and behavior of mated females. In many insect species, and especially in the moth Agrotis ipsilon, juvenile hormone (JH) promotes the maturation of the MAGs but the underlying molecular mechanisms in this hormonal regulation are not yet well identified. Here, we examined the role of the JH receptor, Methoprene-tolerant (Met) and the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) in transmitting the JH signal that upregulates the growth and synthetic activity of the MAGs in A. ipsilon. We cloned two full length cDNAs encoding Met1 and Met2 which are co-expressed with Kr-h1 in the MAGs where their expression levels increase with age in parallel with the length and protein content of the MAGs. RNAi-mediated knockdown of either Met1, Met2, or Kr-h1 resulted in reduced MAG length and protein amount. Moreover, injection of JH-II into newly emerged adult males induced the transcription of Met1, Met2 and Kr-h1 associated to an increase in the length and protein content of the MAGs. By contrast, JH deficiency decreased Met1, Met2 and Kr-h1 mRNA levels as well as the length and protein reserves of the MAGs of allatectomized old males and these declines were partly compensated by a combined injection of JH-II in operated males. Taken together, our results highlighted an involvement of the JH-Met-Kr-h1 signaling pathway in the development and secretory activity of the MAGs in A. ipsilon.

A critical role for Dop1-mediated dopaminergic signaling in the plasticity of behavioral and neuronal responses to sex pheromone in a moth.

Most animal species, including insects, are able to modulate their responses to sexual chemosignals and this flexibility originates from the remodeling of olfactory areas under the influence of the dopaminergic system. In the moth Agrotis ipsilon, the behavioral response of males to the female-emitted sex pheromone increases throughout adult life and after a prior exposure to pheromone signal, and this change is accompanied by an increase in neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs). To identify the underlying neuromodulatory mechanisms, we examined whether this age- and experience-dependent olfactory plasticity is mediated by dopamine (DA) through the Dop1 receptor, an ortholog of the vertebrate D1-type dopamine receptors, which is positively coupled to adenylyl cyclase. We cloned A. ipsilon Dop1 (AiDop1), which is expressed predominantly in brain and especially in ALs; its knockdown induced a decrease in AL cAMP and altered sex pheromone-orientated flight. The levels of DA, AiDop1 expression and cAMP in ALs increased from the third day of adult life and at 24 and 48 h following pre-exposure to sex pheromone, and the dynamic of these changes correlated with the increased responsiveness to sex pheromone. These results demonstrate that Dop1 is required for the display of male sexual behavior and that age- and experience-related neuronal and behavioral changes are sustained by DA-Dop1 signaling that operates within ALs, probably through cAMP-dependent mechanisms in A. ipsilon Thus, this study expands our understanding of the neuromodulatory mechanisms underlying olfactory plasticity, mechanisms that appear to be highly conserved between insects and mammals.

The insect HR38 nuclear receptor, a member of the NR4A subfamily, is a synchronizer of reproductive activity in a moth.

In the male moth, Agrotis ipsilon, the behavioural response and neuron sensitivity within the olfactory centres, the antennal lobes (ALs), to female sex pheromone increase with age, in correlation with the maturation of sex accessory glands (SAGs). By contrast, newly mated males cease to be attracted to sex pheromone and remate when their SAGs are refilled during the next night. The insect hormone receptor 38 (HR38), an ortholog of the vertebrate NR4A receptors, is a component of ecdysteroid signalling pathway which controls adult male physiology and behaviour. Here, we cloned the A. ipsilon HR38 (AiHR38) and explored its function in the coordination of reproductive events in the male. AiHR38 was detected in SAGs and ALs, and where its amount raised with age, in parallel with SAG protein content and sex pheromone responsiveness. By contrast, the AL and SAG AiHR38 expressions declined at 0-2 h after mating, in linking with depletion of SAG protein reserves and loss of sensitivity to sex pheromone. The increased AL and SAG AiHR38 expressions at 20-24 h postmating coincided with replenishing of SAGs and recovery of sensitivity to sex pheromone for a new mating. Moreover, AiHR38 knockdown resulted in reduction in SAG protein amount and disruption of sex pheromone-orientated flight. These results show that the insect HR38 is essential both for SAG activity, probably by controlling the protein synthesis, and display of male sexual behaviour, and that the concomitant regulation of its expression within SAGs and olfactory centres contributes to synchronisation between fertility and sexual activity. DATABASE: The nucleotide sequence of Agrotis ipsilon HR38 is available in the DDBJ/EMBL/GenBank databases under the accession number MF402845.