Hepatocellular carcinoma tumour burden score to stratify prognosis after resection.

BACKGROUND: Although the Barcelona Clinic Liver Cancer (BCLC) staging system has been largely adopted in clinical practice, recent studies have emphasized the need for further refinement and subclassification of this system. METHODS: Patients who underwent hepatectomy with curative intent for BCLC-0, -A or -B hepatocellular carcinoma (HCC) between 2000 and 2017 were identified using a multi-institutional database. The tumour burden score (TBS) was calculated, and overall survival (OS) was examined in relation to TBS and BCLC stage. RESULTS: Among 1053 patients, 63 (6·0 per cent) had BCLC-0, 826 (78·4 per cent) BCLC-A and 164 (15·6 per cent) had BCLC-B HCC. OS worsened incrementally with higher TBS (5-year OS 77·9, 61 and 39 per cent for low, medium and high TBS respectively; P < 0·001). No differences in OS were noted among patients with similar TBS, irrespective of BCLC stage (61·6 versus 58·9 per cent for BCLC-A/medium TBS versus BCLC-B/medium TBS, P = 0·930; 45 versus 13 per cent for BCLC-A/high TBS versus BCLC-B/high TBS, P = 0·175). Patients with BCLC-B HCC and a medium TBS had better OS than those with BCLC-A disease and a high TBS (58·9 versus 45 per cent; P = 0·005). On multivariable analysis, TBS remained associated with OS among patients with BCLC-A (medium TBS: hazard ratio (HR) 2·07, 95 per cent c.i. 1·42 to 3·02, P < 0·001; high TBS: HR 4·05, 2·40 to 6·82, P < 0·001) and BCLC-B (high TBS: HR 3·85, 2·03 to 7·30; P < 0·001) HCC. TBS could also stratify prognosis among patients in an external validation cohort (5-year OS 79, 51·2 and 28 per cent for low, medium and high TBS respectively; P = 0·010). CONCLUSION: The prognosis of patients with HCC varied according to the BCLC stage but was largely dependent on the TBS.

ANTECEDENTES: Aunque el sistema de estadificación del Barcelona Clinic Liver Cancer (BCLC) ha sido adoptado en gran medida en la práctica clínica, estudios recientes han enfatizado la necesidad de un mayor refinamiento y subclasificación del sistema BCLC. MÉTODOS: Los pacientes con carcinoma hepatocelular (hepatocellular cancer, HCC) BCLC-0, A y B que se sometieron a una hepatectomía con intención curativa entre 2000 y 2017 fueron identificados utilizando una base de datos multi-institucional. Se calculó la puntuación de carga tumoral (tumour burden score, TBS) y se examinó la supervivencia global (overall survival, OS) en relación con la TBS y los estadios BCLC. RESULTADOS: En la serie de 1.053 pacientes, 63 (6%) tenían HCC BCLC-0, 826 (78,4%) HCC BCLC-A y 164 (15,6%) HCC BCLC-B. La OS disminuyó de forma incremental en función de la mayor TBS (OS a 5 años; TBS baja: 77,9% versus TBS media: 61% versus TBS alta: 39%, P < 0,001). No se observaron diferencias en la OS entre pacientes con una puntuación TBS similar, independientemente del estadio BCLC (BCLC-A/TBS media: 61,6% versus BCLC-B/TBS media: 58,9%, P = 0,93; BCLC-A/TBS alta: 45,1% versus BCLC-B/TBS alta: 12,8%, P = 0,175). Los pacientes con BCLC-B/TBS media tuvieron una mejor OS que los pacientes con BCLC-A/TBS alta (58,9% versus 45,1%, P = 0,005). En el análisis multivariable, la TBS se mantuvo asociada a la OS en el caso de BCLC-A (TBS media: cociente de riesgos instantáneos, hazard ratio, HR = 2,07, i.c. del 95%: 1,42-3,02, P < 0,001; TBS alta: HR = 4,05, i.c. del 95%: 2,40-6,82, P < 0,001) y BCLC-B pacientes (TBS alta: HR = 3,85, i.c. del 95%: 2,03-7,30, P < 0,001). La TBS también pudo estratificar el pronóstico entre pacientes en una cohorte de validación externa (OS a 5 años; TBS baja: 78,7% versus TBS media: 51,2% versus TBS alta: 27,6%, P = 0,01). CONCLUSIÓN: El pronóstico de los pacientes con HCC varió según el estadio BCLC, pero dependió en gran medida de la TBS.

Activity and inactivity of moth sex chromosomes in somatic and meiotic cells.

Moths and butterflies (Lepidoptera) are the most species-rich group of animals with female heterogamety, females mostly having a WZ, males a ZZ sex chromosome constitution. We studied chromatin conformation, activity, and inactivity of the sex chromosomes in the flour moth Ephestia kuehniella and the silkworm Bombyx mori, using immunostaining with anti-H3K9me2/3, anti-RNA polymerase II, and fluoro-uridine (FU) labelling of nascent transcripts, with conventional widefield fluorescence microscopy and 'spatial structured illumination microscopy' (3D-SIM). The Z chromosome is euchromatic in somatic cells and throughout meiosis. It is transcriptionally active in somatic cells and in the postpachaytene stage of meiosis. The W chromosome in contrast is heterochromatic in somatic cells as well as in meiotic cells at pachytene, but euchromatic and transcriptionally active like all other chromosomes at postpachytene. As the W chromosomes are apparently devoid of protein-coding genes, their transcripts must be non-coding. We found no indication of 'meiotic sex chromosome inactivation' (MSCI) in the two species.

The fate of W chromosomes in hybrids between wild silkmoths, Samia cynthia ssp.: no role in sex determination and reproduction.

Moths and butterflies (Lepidoptera) have sex chromosome systems with female heterogamety (WZ/ZZ or derived variants). The maternally inherited W chromosome is known to determine female sex in the silkworm, Bombyx mori. However, little is known about the role of W chromosome in other lepidopteran species. Here we describe two forms of the W chromosome, W and neo-W, that are transmitted to both sexes in offspring of hybrids from reciprocal crosses between subspecies of wild silkmoths, Samia cynthia. We performed crosses between S. c. pryeri (2n=28, WZ/ZZ) and S. c. walkeri (2n=26, neo-Wneo-Z/neo-Zneo-Z) and examined fitness and sex chromosome constitution in their hybrids. The F1 hybrids of both reciprocal crosses had reduced fertility. Fluorescence in situ hybridization revealed not only the expected sex chromosome constitutions in the backcross and F2 hybrids of both sexes but also females without the W (or neo-W) chromosome and males carrying the W (or neo-W) chromosome. Furthermore, crosses between the F2 hybrids revealed no association between the presence or absence of W (or neo-W) chromosome and variations in the hatchability of their eggs. Our results clearly suggest that the W (or neo-W) chromosome of S. cynthia ssp. plays no role in sex determination and reproduction, and thus does not contribute to the formation of reproductive barriers between different subspecies.

A FISH-based chromosome map for the European corn borer yields insights into ancient chromosomal fusions in the silkworm.

A significant feature of the genomes of Lepidoptera, butterflies and moths, is the high conservation of chromosome organization. Recent remarkable progress in genome sequencing of Lepidoptera has revealed that syntenic gene order is extensively conserved across phylogenetically distant species. The ancestral karyotype of Lepidoptera is thought to be n=31; however, that of the most well-studied moth, Bombyx mori, is n=28, and diverse studies suggest that three chromosomal fusion events occurred in this lineage. To identify the boundaries between predicted ancient fusions involving B. mori chromosomes 11, 23 and 24, we constructed fluorescence in situ hybridization (FISH)-based chromosome maps of the European corn borer, Ostrinia nubilalis (n=31). We first determined a 511 Mb genomic sequence of the Asian corn borer, O. furnacalis, a congener of O. nubilalis, and isolated bacterial artificial chromosomes and fosmid clones that were expected to localize in candidate regions for the boundaries using these sequences. Combined with FISH and genetic analysis, we narrowed down the candidate regions to 40 kb-1.5 Mb, in strong agreement with a previous estimate based on the genome of a butterfly, Melitaea cinxia. The significant difference in the lengths of the candidate regions where no functional genes were observed may reflect the evolutionary time after fusion events.

Mapping and recombination analysis of two moth colour mutations, Black moth and Wild wing spot, in the silkworm Bombyx mori.

Many lepidopteran insects exhibit body colour variations, where the high phenotypic diversity observed in the wings and bodies of adults provides opportunities for studying adaptive morphological evolution. In the silkworm Bombyx mori, two genes responsible for moth colour mutation, Bm and Ws, have been mapped to 0.0 and 14.7 cM of the B. mori genetic linkage group 17; however, these genes have not been identified at the molecular level. We performed positional cloning of both genes to elucidate the molecular mechanisms that underlie the moth wing- and body-colour patterns in B. mori. We successfully narrowed down Bm and Ws to ~2-Mb-long and 100-kb-long regions on the same scaffold Bm_scaf33. Gene prediction analysis of this region identified 77 candidate genes in the Bm region, whereas there were no candidate genes in the Ws region. Fluorescence in-situ hybridisation analysis in Bm mutant detected chromosome inversion, which explains why there are no recombination in the corresponding region. The comparative genomic analysis demonstrated that the candidate regions of both genes shared synteny with a region associated with wing- and body-colour variations in other lepidopteran species including Biston betularia and Heliconius butterflies. These results suggest that the genes responsible for wing and body colour in B. mori may be associated with similar genes in other Lepidoptera.

Step-by-step evolution of neo-sex chromosomes in geographical populations of wild silkmoths, Samia cynthia ssp.

Geographical subspecies of wild silkmoths, Samia cynthia ssp. (Lepidoptera: Saturniidae), differ considerably in sex chromosome constitution owing to sex chromosome fusions with autosomes, which leads to variation in chromosome numbers. We cloned S. cynthia orthologues of 16 Bombyx mori genes and mapped them to chromosome spreads of S. cynthia subspecies by fluorescence in situ hybridization (FISH) to determine the origin of S. cynthia neo-sex chromosomes. FISH mapping revealed that the Z chromosome and chromosome 12 of B. mori correspond to the Z chromosome and an autosome (A1) of S. c. ricini (Vietnam population, 2n=27, Z0 in female moths), respectively. B. mori chromosome 11 corresponds partly to another autosome (A2) and partly to a chromosome carrying nucleolar organizer region (NOR) of this subspecies. The NOR chromosome of S. c. ricini is also partly homologous to B. mori chromosome 24. Furthermore, our results revealed that two A1 homologues each fused with the W and Z chromosomes in a common ancestor of both Japanese subspecies S. c. walkeri (Sapporo population, 2n=26, neo-Wneo-Z) and S. cynthia subsp. indet. (Nagano population, 2n=25, neo-WZ1Z2). One homologue, corresponding to the A2 autosome in S. c. ricini and S. c. walkeri, fused with the W chromosome in S. cynthia subsp. indet. Consequently, the other homologue became a Z2 chromosome. These results clearly showed a step-by-step evolution of the neo-sex chromosomes by repeated autosome-sex chromosome fusions. We suggest that the rearrangements of sex chromosomes may facilitate divergence of S. cynthia subspecies towards speciation.

Ecdysone response element in a baculovirus immediate-early gene, ie1, promoter.

A computer-assisted analysis identified tentative target sequences for regulatory proteins including ecdysone-inducible factors such as BmFTZ-F1 and Broad-Complex Z4 (BR-C Z4) in the ie1 promoter of BmNPV. A transient expression experiment using BmN cells and a series of truncated ie1 promoter constructs demonstrated that the activity of the ie1 promoter responded to alpha-ecdysone and 20-hydroxyecdysone, which required a tridecameric nucleotide stretch (ie1EcRE, 5'-GTGTTATCGACCT-3') homologous to the ecdysone response element reported for Drosophila (DmEcRE). RT-PCR demonstrated the expression of BmEcR and BmUSP, which are required as ecdysone-specific activators for EcRE-mediated activation, in BmN cells. Furthermore, the ie1 EcRE-mediated response was confirmed by using a recombinant BmNPV possessing a luciferase gene under the control of the ie1 promoter with or without ie1 EcRE. This is the first report of an ecdysone response element in a baculoviral gene promoter. These results also suggested that the regulation of the ie1 by ecdysone may militate viral replication at least under certain conditions during natural infections in vivo.

Sex chromosomes and sex determination in Lepidoptera.

The speciose insect order Lepidoptera (moths and butterflies) and their closest relatives, Trichoptera (caddis flies), share a female-heterogametic sex chromosome system. Originally a Z/ZZ (female/male) system, it evolved by chromosome rearrangement to a WZ/ZZ (female/male) system in the most species-rich branch of Lepidoptera, a monophyletic group consisting of Ditrysia and Tischeriina, which together comprise more than 98% of all species. Further sporadic rearrangements created multi-sex chromosome systems; sporadic losses of the W changed the system formally back to Z/ZZ in some species. Primary sex determination depends on a Z-counting mechanism in Z/ZZ species, but on a female-determining gene, Fem, in the W chromosome of the silkworm. The molecular mechanism is unknown in both cases. The silkworm shares the last step, dsx, of the hierarchical sex-determining pathway with Drosophila and other insects investigated, but probably not the intermediate steps between the primary signal and dsx. The W chromosome is heterochromatic in most species. It contains few genes and is flooded with interspersed repetitive elements. In interphase nuclei of females it is readily discernible as a heterochromatic body which grows with increasing degree of polyploidy in somatic cells. It is used as a marker for the genetic sex in studies of intersexes and Wolbachia infections. The sex chromosome system is being exploited in economically important species. Special strains have been devised for mass rearing of male-only broods in the silkworm for higher silk production and in pest species for the release of sterile males in pest management programs.

Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori.

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.

Use of RNAi technology to confer enhanced resistance to BmNPV on transgenic silkworms.

dsRNA is a powerful tool for gene-specific silencing in plants and animals. In this study, we examined the use of gene silencing in generating transgenic silkworms resistant to the Bombyx mori nucleopolyhedrovirus (BmNPV). Using a transposon piggyBac system, we first generated BmN cells (rBmN-lef1), which carried artificial genes designed for expressing dsRNAs with sequences of the essential viral gene lef-1. NPV DNA microarray analysis revealed that the accumulation of lef-1 mRNA was successfully inhibited in rBmN-lef1 infected with BmNPV. The virus titer in the culture medium of rBmN-lef1 at 48 hr post-infection (h.p.i.) was 50% of that of the control cells. Moderate BmNPV-resistance caused by transgenesis of the artificial dsRNA-expressing gene was confirmed in the transgenic silkworms. Virus production was reduced in transgenic silkworms relative to controls up to 96 hrs after viral inoculation. Although complete protection was not achieved and the transgenic larvae ultimately died, this is the first report to show the use of RNAi in confering enhanced viral resistance on transgenic animals.

Glucose and ecdysteroid increase apyrene sperm production in in vitro cultivation of spermatocysts of Bombyx mori.

Two types of sperm, nucleate eupyrene and anucleate apyrene, occur in the silkworm as in other lepidopteran species. Hormones and other substances have been assumed to play important roles in sperm dimorphism. We established an in vitro cultivation system for silkworm spermatocytes, and found that apyrene sperm are not produced when spermatocytes from larval testes are cultivated, though eupyrene spermatocytes develop normally into mature sperm. Based on the fact that ecdysteroid titers increase rapidly and peak 1 day after spinning, and that the amount of glycogen reaches its peak 1 day before the spinning stage, we studied the effects of adding glucose and/or 20-hydroxyecdysone to the culture medium. The experiments disclosed a significant additive effect of both substances on apyrene sperm production.

Significance of peristaltic squeezing of sperm bundles in the silkworm, Bombyx mori: elimination of irregular eupyrene sperm nuclei of the triploid.

Silkworm (Lepidoptera) males produce dimorphic sperm: nucleate eupyrene sperm and anucleate apyrene sperm. The eupyrene sperm are ordinary sperm to fertilise the eggs, while the function of apyrene sperm remains uncertain. After meiosis, 256 sperm cells are enclosed by a layer of cyst cells, forming a sperm bundle. We have previously documented that the nucleus of eupyrene sperm anchors to the head cyst cell, which locates at the anterior apex of the bundle, by an acrosome tubule-basal body assembly. Neither the basal body attachment to the nucleus nor the acrosome is seen in apyrene sperm, and the nuclei remain in the middle region of the bundle. Peristaltic squeezing starts from the anterior of the bundles in both types of sperm, and cytoplasmic debris of the eupyrene sperm, and both the nuclei and debris of apyrene sperm, are eliminated at the final stage of spermatogenesis. Since the irregularity of meiotic division in apyrene sperm is known, we used triploid silkworm males that show irregular meiotic division even in eupyrene spermatocytes and are highly sterile. The irregular nuclei of the triploid are discarded by the peristaltic squeezing just as those of the apyrene sperm. Transmission electron microscopic observations disclose the abnormality in the acrosome tubule and in the connection to the basal body. The peristaltic squeezing of sperm bundles in the silkworm appears to be the final control mechanism to eliminate irregular nuclei before they enter female reproductive organs.

Meiotic pairing of sex chromosome fragments and its relation to atypical transmission of a sex-linked marker in Ephestia kuehniella (Insecta: Lepidoptera).

The physical basis of non-Mendelian segregation of a sex-linked marker was studied in sex- chromosome mutant females of eight ASF ('abnormal segregating females') lines in the flour moth, Ephestia kuehniella. Electron microscopical analysis of microspread synaptonemal complexes revealed that in one line, the Z chromosome segment that contained the dz+ allele was translocated onto an autosome. The resulting quadrivalent visible in early female meiosis was 'corrected' into two bivalents in later stages. This explains autosomal inheritance of the sex chromosome marker in this strain. In the other seven ASF lines, the type of meiotic pairing of an additional fragment (Zdz+) of the Z chromosome was responsible for abnormal segregation of the marker gene. In several of these lines, Zdz+ contained a piece of the W chromosome in addition to the Z segment, as was confirmed by comparative genomic hybridization (CGH). Zdz+ formed three alternative pairing configurations with the original sex chromosomes: (i) a WZZdz+ trivalent, (ii) a WZ bivalent and a Zdz+ univalent or (iii) a ZZdz+ bivalent and a W univalent. In the most frequent WZZdz+ configuration, Zdz+ synapsed with Z and, consequently, segregated with W, simulating W linkage. This explains the predominant occurrence of the parental phenotypes in the progeny. Zdz+ univalents or W univalents, on the other hand, segregated randomly, resulting in both parental and nonparental phenotypes. In two of these lines, the Zdz+ was transmitted only to females. The results suggest that the W chromosome segment in Zdz+ of these lines contains a male-killing factor which makes it incompatible with male development. Our data provide direct evidence for the regular transmission of radiation-induced fragments from lepidopteran chromosomes through more than 50 generations. This is facilitated by the holokinetic nature of lepidopteran chromosomes. We conclude that Zdz+ fragments may persist as long as they possess active kinetochore elements.

Peristaltic squeezing of sperm bundles at the late stage of spermatogenesis in the silkworm, Bombyx mori.

Silkworm (Lepidoptera) males produce dimorphic sperm, nucleate eupyrene sperm, and anucleate apyrene sperm. The eupyrene sperm is the ordinary sperm fertilizing eggs, while the function of the apyrene sperm, which are about four times as numerous as the eupyrene sperm, is still uncertain. We found the peristaltic phenomenon at the very late stage of spermatogenesis. Peristalsis occurs in both eupyrene and apyrene sperm bundles. Through peristaltic action, cytoplasm of the eupyrene sperm and both cytoplasm and nuclei of the apyrene sperm are discarded from the posterior end of the sperm bundles. Peristaltic squeezing seems to be a process to eliminate the irregular nuclei of apyrene sperm while preserving the nuclei of eupyrene sperm.

[Epidemiological analysis of influenza B virus belonging to B/Victoria/2/87 lineage isolated in off-season of 1998 and late epidemic season in Shizuoka Prefecture].

In Shizuoka Prefecture, influenza B viruses belonging to B/Victoria/2/87 lineage caused an outbreak among school children in the off-season of 1998, and the same B viruses were mainly isolated during the following epidemic. Low titer of HI antibody among children for influenza B virus belonging to the same lineage was a recognized factor in the causation of the distinctive epidemic. The herald virus strains seemed to be antigenically similar to the late epidemic virus strains, but the former strains were not genetically close to the latter. This indicates that the herald viruses are not always the parental viruses for the following influenza season.

Analysis of proteins encoded in the bipartite genome of a new type of parvo-like virus isolated from silkworm - structural protein with DNA polymerase motif.

Bombyx mori densonucleosis virus type 2 (BmDNV-2) is a small, spherical virus containing two complementary single-stranded linear DNA molecules (VD1, VD2). BmDNV-2 is a new type of virus with a unique, yet unspecified replication mechanism which is different from that of parvoviruses (Bando, H., Choi, H., Ito, Y., Nakagaki, M. , Kawase, S., 1992. Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence, Arch. Virol. 124, 187-193; Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M. , Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Hayakawa, T., Asano, S., Sahara, K., Iizuka, T., Bando, H., 1997. Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus. Arch. Virol. 142, 1-7). Recent analyses on the genomic information of BmDNV-2 identified open reading frames which code for three tentative nonstructural proteins and four (VP1 to 4) of the six known structural proteins (Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M., Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Nakagaki et al., in preparation). In this report we demonstrate that the two largest ORFs, VD1-ORF1 and VD2-ORF1, code for the two remaining structural proteins. In addition, computer-assisted analysis revealed that the structural protein encoded in VD1-ORF1 contains sequences conserved among various DNA polymerases, and showed an evolutionary relationship with the DNA polymerases involved in protein-primed replication.

TTAGG telomeric repeats in chromosomes of some insects and other arthropods.

We studied the occurrence of the TTAGG telomere repeats by fluorescence in-situ hybridization (FISH) and Southern hybridization in ten insect species and two other arthropods. (TTAGG)n-containing telomeres were found in three Lepidoptera species, the silkworm Bombyx mori (in which the telomeric sequence was recently discovered), the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella, in one species of Hymenoptera, the honey bee Apis mellifera, in one species of Coleoptera, the bark beetle Ips typographus, in one species of Orthoptera, the locust Locusta migratoria, and in a crustacean, the amphipod Gammarus pulex. They were absent in another species of Coleoptera, the mealworm Tenebrio molitor, two representatives of Diptera, Drosophila melanogaster and Megaselia scalaris, a species of Heteroptera, the bug Pyrrhocoris apterus and a spider, Tegenaria ferruginea. Our results, which confirm and extend earlier observations, suggest that (TTAGG)n was a phylogenetically ancestral telomere motif in the insect lineage but was lost independently in different groups, being replaced probably by other telomere motifs. In the Coleoptera this must have happened rather recently as even members of the same family, Curculionidae, differ with respect to the telomeric DNA.

Molecular differentiation of sex chromosomes probed by comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to identify and probe sex chromosomes in several XY and WZ systems. Chromosomes were hybridized simultaneously with FluorX-labelled DNA of females and Cy3-labelled DNA of males in the presence of an excess of Cot-1 DNA or unlabelled DNA of the homogametic sex. CGH visualized the molecular differentiation of the X and Y in the house mouse, Mus musculus, and in Drosophila melanogaster: while autosomes were stained equally by both probes, the X and Y chromosomes were stained preferentially by the female-derived or the male-derived probe, respectively. There was no differential staining of the X and Y chromosomes in the fly Megaselia scalaris, indicating an early stage of sex chromosome differentiation in this species. In the human and the house mouse, labelled DNA of males in the presence of unlabelled DNA of females was sufficient to highlight Y chromosomes in mitosis and interphase. In WZ sex chromosome systems, the silkworm Bombyx mori, the flour moth Ephestia kuehniella, and the wax moth Galleria mellonella, the W chromosomes were identified by CGH in mitosis and meiosis. They were conspicuously stained by both female- and male-derived probes, unlike the Z chromosomes, which were preferentially stained by the male-derived probe in E. kuehniella only but were otherwise inconspicuous. The ratio of female:male staining and the pattern of staining along the W chromosomes was species specific. CGH shows that W chromosomes in these species are molecularly well differentiated from the Z chromosomes. The conspicuous binding of the male-derived probe to the W chromosomes is presumably due to an accumulation of common interspersed repetitive sequences.