Zygosity-based sex determination in a butterfly drives hypervariability of Masculinizer.

Nature has devised many ways of producing males and females. Here, we report on a previously undescribed mechanism for Lepidoptera that functions without a female-specific gene. The number of alleles or allele heterozygosity in a single Z-linked gene (BaMasc) is the primary sex-determining switch in Bicyclus anynana butterflies. Embryos carrying a single BaMasc allele develop into WZ (or Z0) females, those carrying two distinct alleles develop into ZZ males, while (ZZ) homozygotes initiate female development, have mismatched dosage compensation, and die as embryos. Consequently, selection against homozygotes has favored the evolution of spectacular allelic diversity: 205 different coding sequences of BaMasc were detected in a sample of 246 females. The structural similarity of a hypervariable region (HVR) in BaMasc to the HVR in Apis mellifera csd suggests molecular convergence between deeply diverged insect lineages. Our discovery of this primary switch highlights the fascinating diversity of sex-determining mechanisms and underlying evolutionary drivers.

Chromosome analysis and the occurrence of B chromosomes in fish parasite Acanthocephalus anguillae (Palaeacanthocephala: Echinorhynchida).

The cytogenetics of Acanthocephala is a neglected area in the study of this group of endoparasites. Chromosome number and/or karyotypes are known for only 12 of the 1,270 described species, and molecular cytogenetic data are limited to rDNA mapping in two species. The standard karyological technique and mapping of 18S rRNA and H3 histone genes on the chromosomes of Acanthocephalus anguillae individuals from three populations, one of which originated from the unfavorable environmental conditions of the Zemplínska Sírava reservoir in eastern Slovakia, were applied for the first time. All specimens had 2n = 7/8 (male/female); n = 1m + 1m-sm + 1a + 1a (X). Fluorescence in situ hybridization (FISH) revealed three loci of 18S rDNA on two autosomes and dispersion of H3 histone genes on all autosomes and the X chromosome. In addition to the standard A chromosome set, 34% of specimens from Zemplínska Sírava possessed a small acrocentric B chromosome, which was always found to be univalent, with no pairing observed between the B chromosome and the A complement. The B chromosome had a small amount of heterochromatin in the centromeric and telomeric regions of the chromosomal arms and showed two clusters of H3 genes. It is well known that an environment permanently polluted with chemicals leads to an increased incidence of chromosomal rearrangements. As a possible scenario for the B chromosome origin, we propose chromosomal breaks due to the mutagenic effect of pollutants in the aquatic environment. The results are discussed in comparison with previous chromosome data from Echinorhynchida species.

Title: Analyse chromosomique et présence de chromosomes B chez le parasite de poisson Acanthocephalus anguillae (Palaeacanthocephala, Echinorhynchida). Abstract: La cytogénétique des Acanthocephala est un domaine négligé dans l'étude de ce groupe d'endoparasites. Le nombre de chromosomes et/ou les caryotypes ne sont connus que pour 12 des 1270 espèces décrites, et les données cytogénétiques moléculaires se limitent à la cartographie de l'ADNr chez deux espèces. La technique caryologique standard et la cartographie des gènes de l'ARNr 18S et de l'histone H3 ont été appliquées pour la première fois sur les chromosomes d'individus d'Acanthocephalus anguillae provenant de trois populations, dont l'une dans les conditions environnementales défavorables du réservoir de Zemplínska Sírava dans l'est de la Slovaquie. Tous les spécimens avaient 2n = 7/8 (mâle/femelle); n = 1m + 1m-sm + 1a + 1a (X). La technique FISH a révélé trois locus d'ADNr 18S sur deux autosomes et une dispersion des gènes de l'histone H3 sur tous les autosomes et sur le chromosome X. En plus de l'ensemble standard de chromosomes A, 34 % des spécimens de Zemplínska Sírava possédaient un petit chromosome B acrocentrique, qui s'est toujours révélé univalent, sans aucun appariement observé entre le chromosome B et le complément A. Le chromosome B avait une petite quantité d'hétérochromatine dans les régions centromériques et télomériques des bras chromosomiques et présentait deux groupes de gènes H3. Il est bien connu qu'un environnement pollué en permanence par des produits chimiques entraîne une incidence accrue de réarrangements chromosomiques. Comme scénario possible pour l'origine du chromosome B, nous proposons des cassures chromosomiques dues à l'effet mutagène des polluants du milieu aquatique. Les résultats sont discutés en comparaison avec les données chromosomiques précédentes des espèces d'Echinorhynchida.

Development of a novel genetic sexing strain of Ceratitis capitata based on an X-autosome translocation.

Genetic sexing strains (GSS), such as the Ceratitis capitata (medfly) VIENNA 8 strain, facilitate male-only releases and improve the efficiency and cost-effectiveness of sterile insect technique (SIT) applications. Laboratory domestication may reduce their genetic diversity and mating behaviour and hence, refreshment with wild genetic material is frequently needed. As wild males do not carry the T(Y;A) translocation, and wild females do not easily conform to artificial oviposition, the genetic refreshment of this GSS is a challenging and time-consuming process. In the present study, we report the development of a novel medfly GSS, which is based on a viable homozygous T(XX;AA) translocation using the same selectable markers, the white pupae and temperature-sensitive lethal genes. This allows the en masse cross of T(XX;AA) females with wild males, and the backcrossing of F1 males with the T(XX;AA) females thus facilitating the re-establishment of the GSS as well as its genetic refreshment. The rearing efficiency and mating competitiveness of the novel GSS are similar to those of the T(Y;A)-based VIENNA 8 GSS. However, its advantage to easily allow the genetic refreshment is of great importance as it can ensure the mass production of high-quality males and enhanced efficacy of operational SIT programs.

High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae).

BACKGROUND: Morphological and traditional genetic studies of the young Pliocene genus Hyles have led to the understanding that despite its importance for taxonomy, phenotypic similarity of wing patterns does not correlate with phylogenetic relationship. To gain insights into various aspects of speciation in the Spurge Hawkmoth (Hyles euphorbiae), we assembled a chromosome-level genome and investigated some of its characteristics. RESULTS: The genome of a male H. euphorbiae was sequenced using PacBio and Hi-C data, yielding a 504 Mb assembly (scaffold N50 of 18.2 Mb) with 99.9% of data represented by the 29 largest scaffolds forming the haploid chromosome set. Consistent with this, FISH analysis of the karyotype revealed n = 29 chromosomes and a WZ/ZZ (female/male) sex chromosome system. Estimates of chromosome length based on the karyotype image provided an additional quality metric of assembled chromosome size. Rescaffolding the published male H. vespertilio genome resulted in a high-quality assembly (651 Mb, scaffold N50 of 22 Mb) with 98% of sequence data in the 29 chromosomes. The larger genome size of H. vespertilio (average 1C DNA value of 562 Mb) was accompanied by a proportional increase in repeats from 45% in H. euphorbiae (measured as 472 Mb) to almost 55% in H. vespertilio. Several wing pattern genes were found on the same chromosomes in the two species, with varying amounts and positions of repetitive elements and inversions possibly corrupting their function. CONCLUSIONS: Our two-fold comparative genomics approach revealed high gene synteny of the Hyles genomes to other Sphingidae and high correspondence to intact Merian elements, the ancestral linkage groups of Lepidoptera, with the exception of three simple fusion events. We propose a standardized approach for genome taxonomy using nucleotide homology via scaffold chaining as the primary tool combined with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements. The identification of wing pattern genes promises future understanding of the evolution of forewing patterns in the genus Hyles, although further sequencing data from more individuals are needed. The genomic data obtained provide additional reliable references for further comparative studies in hawkmoths (Sphingidae).

Masculinizer gene controls male sex determination in the codling moth, Cydia pomonella.

The molecular mechanisms of sex determination in moths and butterflies (Lepidoptera) with female heterogamety (WZ/ZZ) are poorly understood, except in the silkworm Bombyx mori. However, the Masculinizer (Masc) gene that controls male development and dosage compensation in B. mori, appears to be conserved in Lepidoptera, as its masculinizing function was recently confirmed in several moth species. In this work, we investigated the role of the Masc gene in sex determination of the codling moth Cydia pomonella (Tortricidae), a globally important pest of pome fruits and walnuts. The gene structure of the C. pomonella Masc ortholog, CpMasc, is similar to B. mori Masc. However, unlike B. mori, we identified 14 splice variants of CpMasc in the available transcriptomes. Subsequent screening for sex specificity and genetic variation using publicly available data and RT-PCR revealed three male-specific splice variants. Then qPCR analysis of these variants revealed sex-biased expression showing a peak only in early male embryos. Knockdown of CpMasc by RNAi during early embryogenesis resulted in a shift from male-to female-specific splicing of the C. pomonella doublesex (Cpdsx) gene, its downstream effector, in ZZ embryos, leading to a strongly female-biased sex ratio. These data clearly demonstrate that CpMasc functions as a masculinizing gene in the sex-determining cascade of C. pomonella. Our study also showed that CpMasc transcripts are provided maternally, as they were detected in unfertilized eggs after oviposition and in mature eggs dissected from virgin females. This finding is unique, as maternal provision of mRNA has rarely been studied in Lepidoptera.

Deviations in the Z:A ratio disrupt sexual development in the eri silkmoth, Samia cynthia ricini.

Moths and butterflies (Lepidoptera) have sex chromosome systems with female heterogamety, and 2 models, W-dominance and Z-counting, have been proposed to determine sex. The W-dominant mechanism is well known in Bombyx mori. However, little is known about the Z-counting mechanism in Z0/ZZ species. We investigated whether ploidy changes affect sexual development and gene expression in the eri silkmoth, Samia cynthia ricini (2n = 27♀/28♂, Z0♀/ZZ♂). Tetraploid males (4n = 56, ZZZZ) and females (4n = 54, ZZ) were induced by heat and cold shock, and then, triploid embryos were produced by crosses between diploids and tetraploids. Two karyotypes (3n = 42, ZZZ and 3n = 41, ZZ) were identified in triploid embryos. Triploid embryos with 3 Z chromosomes showed male-specific splicing of the S. cynthia doublesex (Scdsx) gene, whereas 2-Z triploid embryos showed both male- and female-specific splicing. From larva to adult, 3-Z triploids showed a normal male phenotype, except for defects in spermatogenesis. However, abnormal gonads were observed in 2-Z triploids, which showed both male- and female-specific Scdsx transcripts not only in the gonads but also in somatic tissues. Two-Z triploids were thus obviously intersexes, suggesting that sexual development in S. c. ricini depends on the Z:A ratio and not only on the Z number. Moreover, mRNA-seq analyses in embryos showed that relative levels of gene expression are similar between samples with different doses of Z chromosomes and autosome sets. Our results provide the first evidence that ploidy changes disrupt sexual development but have no effect on the general mode of dosage compensation in Lepidoptera.

Accumulation of retrotransposons contributes to W chromosome differentiation in the willow beauty Peribatodes rhomboidaria (Lepidoptera: Geometridae).

The W chromosome of Lepidoptera is typically gene-poor, repeat-rich and composed of heterochromatin. Pioneering studies investigating this chromosome reported an abundance of mobile elements. However, the actual composition of the W chromosome varies greatly between species, as repeatedly demonstrated by comparative genomic hybridization (CGH) or fluorescence in situ hybridization (FISH). Here we present an analysis of repeats on the W chromosome in the willow beauty, Peribatodes rhomboidaria (Geometridae), a species in which CGH predicted an abundance of W-enriched or W-specific sequences. Indeed, comparative analysis of male and female genomes using RepeatExplorer identified ten putative W chromosome-enriched repeats, most of which are LTR or LINE mobile elements. We analysed the two most abundant: PRW LINE-like and PRW Bel-Pao. The results of FISH mapping and bioinformatic analysis confirmed their enrichment on the W chromosome, supporting the hypothesis that mobile elements are the driving force of W chromosome differentiation in Lepidoptera. As the W chromosome is highly underrepresented in chromosome-level genome assemblies of Lepidoptera, this recently introduced approach, combining bioinformatic comparative genome analysis with molecular cytogenetics, provides an elegant tool for studying this elusive and rapidly evolving part of the genome.

On the Origin of Neo-Sex Chromosomes in the Neotropical Dragonflies Rhionaeschna bonariensis and R. planaltica (Aeshnidae, Odonata).

Odonata have holokinetic chromosomes. About 95% of species have an XX/X0 sex chromosome system, with heterogametic males. There are species with neo-XX/neo-XY sex chromosomes resulting from an X chromosome/autosome fusion. The genus Rhionaeschna includes 42 species found in the Americas. We analyzed the distribution of the nucleolar organizer region (NOR) using FISH with rDNA probes in Rhionaeschna bonariensis (n = 12 + neo-XY), R. planaltica (n = 7 + neo-XY), and Aeshna cyanea (n = 13 + X0). In R. bonariensis and A. cyanea, the NOR is located on a large pair of autosomes, which have a secondary constriction in the latter species. In R. planaltica, the NOR is located on the ancestral part of the neo-X chromosome. Meiotic analysis and FISH results in R. planaltica led to the conclusion that the neo-XY system arose by insertion of the ancestral X chromosome into an autosome. Genomic in situ hybridization, performed for the first time in Odonata, highlighted the entire neo-Y chromosome in meiosis of R. bonariensis, suggesting that it consists mainly of repetitive DNA. This feature and the terminal chiasma localization suggest an ancient origin of the neo-XY system. Our study provides new information on the origin and evolution of neo-sex chromosomes in Odonata, including new types of chromosomal rearrangements, NOR transposition, and heterochromatin accumulation.

A step forward in the genome characterization of the sugarcane borer, Diatraea saccharalis: karyotype analysis, sex chromosome system and repetitive DNAs through a cytogenomic approach.

Moths of the family Crambidae include a number of pests that cause economic losses to agricultural crops. Despite their economic importance, little is known about their genome architecture and chromosome evolution. Here, we characterized the chromosomes and repetitive DNA of the sugarcane borer Diatraea saccharalis using a combination of low-pass genome sequencing, bioinformatics, and cytogenetic methods, focusing on the sex chromosomes. Diploid chromosome numbers differed between the sexes, i.e., 2n = 33 in females and 2n = 34 in males. This difference was caused by the occurrence of a WZ1Z2 trivalent in female meiosis, indicating a multiple sex-chromosome system WZ1Z2/Z1Z1Z2Z2. A strong interstitial telomeric signal was observed on the W chromosome, indicating a fusion of the ancestral W chromosome with an autosome. Among repetitive DNAs, transposable elements (TEs) accounted for 39.18% (males) to 41.35% (females), while satDNAs accounted for only 0.214% (males) and 0.215% (females) of the genome. FISH mapping revealed different chromosomal organization of satDNAs, such as single localized clusters, spread repeats, and non-clustered repeats. Two TEs mapped by FISH were scattered. Although we found a slight enrichment of some satDNAs in the female genome, they were not differentially enriched on the W chromosome. However, we found enriched FISH signals for TEs on the W chromosome, suggesting their involvement in W chromosome degeneration and differentiation. These data shed light on karyotype and repetitive DNA dynamics due to multiple chromosome fusions in D. saccharalis, contribute to the understanding of genome structure in Lepidoptera and are important for future genomic studies.

New cytogenetic data on Caryophyllaeus laticeps and Paracaryophyllaeus gotoi, parasites of evolutionary interest.

Caryophyllideans are intestinal parasites of freshwater fishes, occupying a basal position among the 'true' tapeworms. We performed detailed cytogenetic analyses of the well-known caryophyllidean species Caryophyllaeus laticeps. For comparison, we also examined for the first time the chromosomes of Paracaryophyllaeus gotoi, a specific parasite of loaches in China. Both species showed a diploid chromosome number of 2n = 20, n = 10m. Chromomycin A3 (CMA3)/diamidino-2-phenylindole (DAPI) staining performed for the first time in the class Cestoda revealed CMA3+/DAPI− bands in the pericentromeric regions of the short arms of chromosome pair no. 7 in the karyotype of C. laticeps. Fluorescence in situ hybridization with the 18S rDNA probe confirmed the presence of a single cluster of major rDNA near the centromere on a pair of small chromosomes in both species. These findings support the hypothesis that the ancestral state in the family Caryophyllaeidae is a single interstitial cluster of major rDNA genes and thus one nucleolar organizer region per haploid genome. Our results, which we presented together with literature data plotted on a phylogenetic tree, show stability of caryophyllidean karyotypes at the genus level, but showed differences between genera without a clear phylogenetic signal. The data allowed us to at least formulate a hypothesis about the ancestral haploid chromosome number of n = 10 for the family Caryophyllaeidae and possibly for the sister family Capingentidae. In addition, we compared two populations of C. laticeps from water bodies with different levels of polychlorinated biphenyl contamination, showing a slightly increased incidence of chromosomal abnormalities at the contaminated site.

Degenerated, Undifferentiated, Rearranged, Lost: High Variability of Sex Chromosomes in Geometridae (Lepidoptera) Identified by Sex Chromatin.

Sex chromatin is a conspicuous body that occurs in polyploid nuclei of most lepidopteran females and consists of numerous copies of the W sex chromosome. It is also a cytogenetic tool used to rapidly assess the W chromosome presence in Lepidoptera. However, certain chromosomal features could disrupt the formation of sex chromatin and lead to the false conclusion that the W chromosome is absent in the respective species. Here we tested the sex chromatin presence in 50 species of Geometridae. In eight selected species with either missing, atypical, or normal sex chromatin patterns, we performed a detailed karyotype analysis by means of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The results showed a high diversity of W chromosomes and clarified the reasons for atypical sex chromatin, including the absence or poor differentiation of W, rearrangements leading to the neo-W emergence, possible association with the nucleolus, and the existence of multiple W chromosomes. In two species, we detected intraspecific variability in the sex chromatin status and sex chromosome constitution. We show that the sex chromatin is not a sufficient marker of the W chromosome presence, but it may be an excellent tool to pinpoint species with atypical sex chromosomes.

A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella.

Sex determination in the silkworm, Bombyx mori, is based on Feminizer (Fem), a W-linked Fem piRNA that triggers female development in WZ individuals, and the Z-linked Masculinizer (Masc), which initiates male development and dosage compensation in ZZ individuals. While Fem piRNA is missing in a close relative of B. mori, Masc determines sex in several representatives of distant lepidopteran lineages. We studied the molecular mechanisms of sex determination in the Mediterranean flour moth, Ephestia kuehniella (Pyralidae). We identified an E. kuehniella Masc ortholog, EkMasc, and its paralog resulting from a recent duplication, EkMascB. Both genes are located on the Z chromosome and encode a similar Masc protein that contains two conserved domains but has lost the conserved double zinc finger domain. We developed PCR-based genetic sexing and demonstrated a peak in the expression of EkMasc and EkMascB genes only in early male embryos. Simultaneous knock-down experiments of both EkMasc and EkMascB using RNAi during early embryogenesis led to a shift from male- to female-specific splicing of the E. kuehniella doublesex gene (Ekdsx), their downstream effector, in ZZ embryos and resulted in a strong female-biased sex-ratio. Our results thus confirmed the conserved role of EkMasc and/or EkMascB in masculinization. We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera. The function of the Masc double zinc finger domain is still unknown, but appears to have been lost in E. kuehniella.

Large-scale comparative analysis of cytogenetic markers across Lepidoptera.

Fluorescence in situ hybridization (FISH) allows identification of particular chromosomes and their rearrangements. Using FISH with signal enhancement via antibody amplification and enzymatically catalysed reporter deposition, we evaluated applicability of universal cytogenetic markers, namely 18S and 5S rDNA genes, U1 and U2 snRNA genes, and histone H3 genes, in the study of the karyotype evolution in moths and butterflies. Major rDNA underwent rather erratic evolution, which does not always reflect chromosomal changes. In contrast, the hybridization pattern of histone H3 genes was well conserved, reflecting the stable organisation of lepidopteran genomes. Unlike 5S rDNA and U1 and U2 snRNA genes which we failed to detect, except for 5S rDNA in a few representatives of early diverging lepidopteran lineages. To explain the negative FISH results, we used quantitative PCR and Southern hybridization to estimate the copy number and organization of the studied genes in selected species. The results suggested that their detection was hampered by long spacers between the genes and/or their scattered distribution. Our results question homology of 5S rDNA and U1 and U2 snRNA loci in comparative studies. We recommend the use of histone H3 in studies of karyotype evolution.

The Role of Satellite DNAs in Genome Architecture and Sex Chromosome Evolution in Crambidae Moths.

Tandem repeats are important parts of eukaryotic genomes being crucial e.g., for centromere and telomere function and chromatin modulation. In Lepidoptera, knowledge of tandem repeats is very limited despite the growing number of sequenced genomes. Here we introduce seven new satellite DNAs (satDNAs), which more than doubles the number of currently known lepidopteran satDNAs. The satDNAs were identified in genomes of three species of Crambidae moths, namely Ostrinia nubilalis, Cydalima perspectalis, and Diatraea postlineella, using graph-based computational pipeline RepeatExplorer. These repeats varied in their abundance and showed high variability within and between species, although some degree of conservation was noted. The satDNAs showed a scattered distribution, often on both autosomes and sex chromosomes, with the exception of both satellites in D. postlineella, in which the satDNAs were located at a single autosomal locus. Three satDNAs were abundant on the W chromosomes of O. nubilalis and C. perspectalis, thus contributing to their differentiation from the Z chromosomes. To provide background for the in situ localization of the satDNAs, we performed a detailed cytogenetic analysis of the karyotypes of all three species. This comparative analysis revealed differences in chromosome number, number and location of rDNA clusters, and molecular differentiation of sex chromosomes.

Molecular cytogenetic analysis of a triploid population of the human broad tapeworm, Dibothriocephalus latus (Diphyllobothriidea).

The large-sized tapeworm Dibothriocephalus latus is known as the broad or fish-borne cestode of mammals that is capable to infect humans and cause diphyllobothriosis. Recently, molecular data on D. latus has been accumulating in the literature and a complete genome sequence has been published; however, little is known about the karyotype and chromosome architecture. In this study, an in-depth karyological analysis of 2 D. latus specimens was carried out. The plerocercoids originated from a perch caught in subalpine Lake Iseo (Italy) and the tapeworms were reared in hamsters. Both specimens contained cells with a highly variable number of chromosomes ranging from18 to 27. Nevertheless, the largest portion of mitotic figures (47%) showed a number corresponding to the triploid set, 3n = 27. Accordingly, the karyotype of the analyzed specimens consisted of 9 triplets of metacentric chromosomes. Fluorescence in situ hybridization (FISH) with the 18S rDNA probe clearly demonstrated the presence of 3 clusters of hybridization signals on the triplet of chromosome 7, thus confirming the triploid status of the specimens. FISH with a telomeric (TTAGGG)n probe confined hybridization signals exclusively to the terminal chromosomal regions, supporting the earlier findings that this repetitive motif is a conserved feature of tapeworm telomeres.

Universal fluorescence in situ hybridization (FISH) protocol for mapping repetitive DNAs in insects and other arthropods.

Repetitive DNAs comprise large portion of eukaryote genomes. In genome projects, the assembly of repetitive DNAs is challenging due to the similarity between repeats, which generate ambiguities for alignment. Fluorescence in situ hybridization (FISH) is a powerful technique for the physical mapping of various sequences on chromosomes. This technique is thus very helpful in chromosome-based genome assemblies, providing information on the fine architecture of genomes and their evolution. However, various protocols are currently used for FISH mapping, most of which are relatively laborious and expensive, or work properly only with a specific type of probes or sequences, and there is a need for a universal and affordable FISH protocol. Here we tested a FISH protocol for mapping of different DNA repeats, such as multigene families (rDNAs, U snDNAs, histone genes), satellite DNAs, microsatellites, transposable elements, DOP-PCR products, and telomeric motif (TTAGG)n, on the chromosomes of various insects and other arthropods. Different cell types and stages obtained from diverse tissues were used. The FISH procedure proved high quality and reliable results in all experiments performed. We obtained data on the chromosomal distribution of DNA repeats in representatives of insects and other arthropods. Thus, our results allow us to conclude that the protocol is universal and requires only time adjustment for chromosome/DNA denaturation. The use of this FISH protocol will facilitate studies focused on understanding the evolution and role of repetitive DNA in arthropod genomes.

White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp- mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp- mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

Patterns of Sex Chromosome Differentiation in Spiders: Insights from Comparative Genomic Hybridisation.

Spiders are an intriguing model to analyse sex chromosome evolution because of their peculiar multiple X chromosome systems. Y chromosomes were considered rare in this group, arising after neo-sex chromosome formation by X chromosome-autosome rearrangements. However, recent findings suggest that Y chromosomes are more common in spiders than previously thought. Besides neo-sex chromosomes, they are also involved in the ancient X1X2Y system of haplogyne spiders, whose origin is unknown. Furthermore, spiders seem to exhibit obligatorily one or two pairs of cryptic homomorphic XY chromosomes (further cryptic sex chromosome pairs, CSCPs), which could represent the ancestral spider sex chromosomes. Here, we analyse the molecular differentiation of particular types of spider Y chromosomes in a representative set of ten species by comparative genomic hybridisation (CGH). We found a high Y chromosome differentiation in haplogyne species with X1X2Y system except for Loxosceles spp. CSCP chromosomes exhibited generally low differentiation. Possible mechanisms and factors behind the observed patterns are discussed. The presence of autosomal regions marked predominantly or exclusively with the male or female probe was also recorded. We attribute this pattern to intraspecific variability in the copy number and distribution of certain repetitive DNAs in spider genomes, pointing thus to the limits of CGH in this arachnid group. In addition, we confirmed nonrandom association of chromosomes belonging to particular CSCPs at spermatogonial mitosis and spermatocyte meiosis and their association with multiple Xs throughout meiosis. Taken together, our data suggest diverse evolutionary pathways of molecular differentiation in different types of spider Y chromosomes.

Evolution of multiple sex-chromosomes associated with dynamic genome reshuffling in Leptidea wood-white butterflies.

Sex-chromosome systems tend to be highly conserved and knowledge about their evolution typically comes from macroevolutionary inference. Rapidly evolving complex sex-chromosome systems represent a rare opportunity to study the mechanisms of sex-chromosome evolution at unprecedented resolution. Three cryptic species of wood-white butterflies-Leptidea juvernica, L. sinapis and L. reali-have each a unique set of multiple sex-chromosomes with 3-4 W and 3-4 Z chromosomes. Using a transcriptome-based microarray for comparative genomic hybridisation (CGH) and a library of bacterial artificial chromosome (BAC) clones, both developed in L. juvernica, we identified Z-linked Leptidea orthologs of Bombyx mori genes and mapped them by fluorescence in situ hybridisation (FISH) with BAC probes on multiple Z chromosomes. In all three species, we determined synteny blocks of autosomal origin and reconstructed the evolution of multiple sex-chromosomes. In addition, we identified W homologues of Z-linked orthologs and characterised their molecular differentiation. Our results suggest that the multiple sex-chromosome system evolved in a common ancestor as a result of dynamic genome reshuffling through repeated rearrangements between the sex chromosomes and autosomes, including translocations, fusions and fissions. Thus, the initial formation of neo-sex chromosomes could not have played a role in reproductive isolation between these Leptidea species. However, the subsequent species-specific fissions of several neo-sex chromosomes could have contributed to their reproductive isolation. Then, significantly increased numbers of Z-linked genes and independent neo-W chromosome degeneration could accelerate the accumulation of genetic incompatibilities between populations and promote their divergence resulting in speciation.