Differential Juvenile Hormone Variations in Scale Insect Extreme Sexual Dimorphism

Abstract

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.

Fig 1. Life cycle of Planococcus krauhniae.

E: Egg, N1: first-instar nymph (undifferentiated in males and females), N2: second-instar nymph (the first few days are undifferentiated), N2♂: male second-instar nymph (identifiable by filamentous secretions), N2♀: female second-instar nymph (filamentous secretions absent), N3: female third-instar nymph, Pre: male prepupa, Pu: male pupa, Ad: adult. Scale bars: 200 μm (E, N1 and N2), 500 μm (N2♀, N2♂, Pre, Pu, Ad). N1, N2, N3, Pre, Pu, and female adult specimens had their secretions removed in the photographs. Arrows: molting events. Red outline: non-feeding stages. Grey gradient: degree of sexual dimorphism.

Fig 2. JH biosynthesis, JH signal transduction, and early responses.

JH biosynthesis, JH signal transduction and early response. Expression profiles of Pkjhamt (A), PkMet (B), Pktai (C), and PkKr-h1 (D) during male and female mealybug development after egg oviposition. E: Egg, N1: first-instar nymph, N2: early second-instar nymphs, N3: female third-instar nymph, f: female adult, pre: male prepupa, pu: male pupa, m: male adult. Samples were collected from E to N2D3, using the sex-bias strategy (see the Methods section).

Fig 3. JHM pyriproxyfen effects on male development and JH signaling.

A. Treatment during prepupa. B. Treatment during pupa. (C-I) Effects of JHM on gene expression. PreD1: Treatment 24-48 hours after the prepupal molt, RNA extraction 5 days after the treatment, N = 6. PuD0: Treatment 0-24 hours after the pupal molt, RNA extraction 6 days after the treatment, N = 5. Boxplots were constructed with the ggplot2 R package (with upper and lower hinges: 1st and 3rd quartiles, middle line: median, dots: outlier values). Unpaired two-sample Student’s t-test with *: p-value < 0.05 and **: p-value < 0.01. C. PkMet; D. Pktai com; E. Pktai IN-1; F. Pktai DEL-1; G. PkKr-h1; H. PkKr-h1 A; I. PkKr-h1 B.

Fig 4. br copies and zinc-finger isoforms in the Japanese mealybug.

A. Protein alignment of the zinc-finger motif for each identified isoform. B. Phylogeny of zinc-finger isoforms for different insect groups. See Supporting Information for the phylogenetic analysis method.

Fig 5. Expression profiles and JHM effects on br.

A. Expression profiles of Pkbr copies identified in the present study. E: Egg, N1: first-instar nymph, N2: second-instar nymphs, N3: female third-instar nymph, Pre: male prepupa, Pu: male pupa. Samples were collected using the sex-bias strategy from E to N2D3. B-F. Effects of JHM on gene expression. PreD1: Treatment 24-48 hours after the prepupal molt, RNA extraction 5 days after the treatment, N = 6. PuD0: treatment 0-24 hours after the pupal molt, RNA extraction 6 days after the treatment, N = 5. Boxplots were constructed with the ggplot2 R package (with upper and lower hinges: 1st and 3rd quartiles, middle line: median, dots: outlier values). Unpaired two-sample Student’s t-test with *: p-value < 0.05 and **: p-value < 0.01. B. Pkbr1 com, C. Pkbr1 Z2, D. Pkbr1 Z4, E. Pkbr3 com, F. Pkbr3 Z2.

Fig 6. Summary of differential JH titers and early response gene patterns when phenotypic differences arise during post-embryonic development in P. kraunhiae.

N2: Second-instar nymph, N3: female third-instar nymph, Pre: prepupa, Pu: pupa.