Sex-specific expression profiles of ecdysteroid biosynthesis and ecdysone response genes in extreme sexual dimorphism of the mealybug Planococcus kraunhiae (Kuwana).

Insect molting hormone (ecdysteroids) and juvenile hormone regulate molting and metamorphic events in a variety of insect species. Mealybugs undergo sexually dimorphic metamorphosis: males develop into winged adults through non-feeding, pupa-like stages called prepupa and pupa, while females emerge as neotenic wingless adults. We previously demonstrated, in the Japanese mealybug Planococcus kraunhiae (Kuwana), that the juvenile hormone titer is higher in males than in females at the end of the juvenile stage, which suggests that juvenile hormone may regulate male-specific adult morphogenesis. Here, we examined the involvement of ecdysteroids in sexually dimorphic metamorphosis. To estimate ecdysteroid titers, quantitative RT-PCR analyses of four Halloween genes encoding for cytochrome P450 monooxygenases in ecdysteroid biosynthesis, i.e., spook, disembodied, shadow and shade, were performed. Overall, their expression levels peaked before each nymphal molt. Transcript levels of spook, disembodied and shadow, genes that catalyze the steps in ecdysteroid biosynthesis in the prothoracic gland, were higher in males from the middle of the second nymphal instar to adult emergence. In contrast, the expression of shade, which was reported to be involved in the conversion of ecdysone into 20-hydroxyecdysone in peripheral tissues, was similar between males and females. These results suggest that ecdysteroid biosynthesis in the prothoracic gland is more active in males than in females, although the final conversion into 20-hydroxyecdysone occurs at similar levels in both sexes. Moreover, expression profiles of ecdysone response genes, ecdysone receptor and ecdysone-induced protein 75B, were also analyzed. Based on these expression profiles, we propose that the changes in ecdysteroid titer differ between males and females, and that high ecdysteroid titer is essential for directing male adult development.

E93 expression and links to the juvenile hormone in hemipteran mealybugs with insights on female neoteny.

Insect metamorphosis produces reproductive adults and is commonly accompanied with the direct or indirect development of wings. In some winged insects, the imago is altered by life history changes. For instance, in scale insects and mealybugs, reproductive females retain juvenile features and are wingless. The transcription factor E93 triggers metamorphosis and plays in concert with the juvenile hormone pathway to guarantee the successful transition from juvenile to adult. We previously provided evidence of an atypical down-regulation of the juvenile hormone pathway during female development in the Japanese mealybug. Here, we further investigate how E93 is involved in the production of neotenic wingless females, by identifying its isoforms, assessing their expression patterns and evaluating the effect of exogenous juvenile hormone mimic treatment on E93. This study identifies three E93 isoforms on the 5' end, based on Japanese mealybug cDNA and shows that female development occurs with the near absence of E93 transcripts, as opposed to male metamorphosis. Additionally, while male development is typically affected by exogenous juvenile hormone mimic treatments, females seem to remain insensitive to the treatment, and up-regulation of the juvenile hormone signaling is not observed. Furthermore, juvenile hormone mimic treatment on female nymphs did not have an obvious effect on E93 transcription, while treatment on male prepupae resulted in depleted E93 transcripts. In this study, we emphasize the importance in examining atypical cases of metamorphosis as complementary systems to provide a better understanding on the molecular mechanisms underlying insect metamorphosis. For instance, the factors regulating the expression of E93 are largely unclear. Investigating the regulatory mechanism of E93 transcription could provide clues towards identifying the factors that induce or suppress E93 transcription, in turn triggering male adult development or female neoteny.

Differential Juvenile Hormone Variations in Scale Insect Extreme Sexual Dimorphism.

Scale insects have evolved extreme sexual dimorphism, as demonstrated by sedentary juvenile-like females and ephemeral winged males. This dimorphism is established during the post-embryonic development; however, the underlying regulatory mechanisms have not yet been examined. We herein assessed the role of juvenile hormone (JH) on the diverging developmental pathways occurring in the male and female Japanese mealybug Planococcus kraunhiae (Kuwana). We provide, for the first time, detailed gene expression profiles related to JH signaling in scale insects. Prior to adult emergence, the transcript levels of JH acid O-methyltransferase, encoding a rate-limiting enzyme in JH biosynthesis, were higher in males than in females, suggesting that JH levels are higher in males. Furthermore, male quiescent pupal-like stages were associated with higher transcript levels of the JH receptor gene, Methoprene-tolerant and its co-activator taiman, as well as the JH early-response genes, Krüppel homolog 1 and broad. The exposure of male juveniles to an ectopic JH mimic prolonged the expression of Krüppel homolog 1 and broad, and delayed adult emergence by producing a supernumeral pupal stage. We propose that male wing development is first induced by up-regulated JH signaling compared to female expression pattern, but a decrease at the end of the prepupal stage is necessary for adult emergence, as evidenced by the JH mimic treatments. Furthermore, wing development seems linked to JH titers as JHM treatments on the pupal stage led to wing deformation. The female pedomorphic appearance was not reflected by the maintenance of high levels of JH. The results in this study suggest that differential variations in JH signaling may be responsible for sex-specific and radically different modes of metamorphosis.