INTRODUCTION In the Lecanoid chromosome system of Coccids, or mealybugs, two important genetic phenomena are widely expressed: facultative heterochromatization and chromosome imprinting. The classical cytogenetic descriptions of Coccids (Hughes- Schrader, 1948; Brown and Nur, 1964) showed that both of these chromosomal processes are developmentally regulated. In the Lecanoid system, chromosomes are holocentric and there are no sex chromosomes. All embryos start development from fertilized eggs and both males and females begin with diploid euchromatic chromosome complements. The paternal and maternal chromosome sets remain euchromatic throughout ontogeny of females in the soma and the germline (Brown and Nur, 1964). By contrast, in embryos that are to become male, the entire paternally derived haploid chromosome set becomes heterochromatic at mid-cleavage. In subsequent cell divisions, the derivatives of the paternal chromosome set appear as chromocenters in interphase nuclei in most tissues. The heterochromatic set retains the cytological properties of condensed metaphase chromosomes throughout the cell cycle. It is distinguishable from the euchromatic one until metaphase, when both sets reach the same degree of condensation. During spermatogenesis, the heterochromatic set is eliminated and only the maternally derived euchromatic chromosomes are included in the sperm. The inactivation of the haploid paternal set of chromosomes represents a dramatic example of facultative heterochromatization that renders male individuals functionally haploid (parahaploidy). Moreover it is evident that there is a mechanism for distinguishing between chromosome sets of maternal and paternal origins (genomic imprinting) in the developing male embryo (Nur, 1990; Bongiorni et al., 1999). Our previous work on imprinting in Coccids (Bongiorni et al., 1999) suggested that the paternal and maternal chromosome sets are characterized by different levels of CpG methylation in embryos. Specifically, we provided cytological evidence for hypomethylation of the paternal set of chromosomes relative to the maternal set. Interestingly, both male and female embryos showed this difference, suggesting that methylation state at a whole chromosomal level might serve as a distinguishing mark of parental origin but it does not reflect overall transcriptional activity of the chromosome sets. We now further investigate the underlying molecular basis for imprinting by asking whether a chromosomal protein, HP-1, which is implicated in heterochromatization in other systems, is involved in imprinting and faculative heterochromatization in Coccids. In 1928, Heitz defined the heterochromatin as regions of chromosomes that do not undergo cyclical changes in condensation during cell cycle as the other chromosome regions (euchromatin) do (Heitz, 1928). The condensed appearance of heterochromatin in higher eukaryotes is thought to be due to a specialized nucleoprotein structure. Thus far, 3809 Development 128, 3809-3817 (2001) Printed in Great Britain © The Company of Biologists Limited 2001 DEV5453 The behavior of chromosomes during development of the mealybug Planococcus citri provides one of the most dramatic examples of facultative heterochromatization. In male embryos, the entire haploid paternal chromosome set becomes heterochromatic at mid-cleavage. Male mealybugs are thus functionally haploid, owing to heterochromatization (parahaploidy). To understand the mechanisms underlying facultative heterochromatization in male mealybugs, we have investigated the possible involvement of an HP-1-like protein in this process. HP-1 is a conserved, nonhistone chromosomal protein with a proposed role in heterochromatinization in other species. It was first identified in Drosophila melanogaster as a protein enriched in the constitutive heterochromatin of polytene chromosome. Using a monoclonal antibody raised against the Drosophila HP-1 in immunoblot and immunocytological experiments, we provide evidence for the presence of an HP-1-like in Planococcus citri males and females. In males, the HP-1-like protein is preferentially associated with the male-specific heterochromatin. In the developing male embryos, its appearance precedes the onset of heterochromatization. In females, the HP-1-like protein displays a scattered but reproducible localization pattern along chromosomes. The results indicate a role for an HP-1-like protein in the facultative heterochromatization process. Key words: Facultative heterochromatization, HP-1, Mealybug SUMMARY Facultative heterochromatization in parahaploid male mealybugs: involvement of a heterochromatin-associated protein Silvia Bongiorni, Milena Mazzuoli, Stefania Masci and Giorgio Prantera* Dipartimento di Agrobiologia e Agrochimica, Università della Tuscia, 01100 Viterbo, Italy *Author for correspondence (e-mail: prantera@unitus.it) Accepted 12 July 2001