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L. cuprina larvae with X-linked or autosomal fluorescent protein transgenes. The lines carry a constitutively expressed DsRed-express2 marker gene. Larvae from an X-linked line (SLAM5) and an autosomal line (EF3C) are shown. In the X-linked line, the most brightly fluorescent larvae develop as females (F). The more weakly fluorescent larvae develop as males (M). In contrast, larvae from the EF3C line show a uniform level of fluorescence. Two representative larvae from this line are shown, the sex of the larvae is unknown.
Source publication
In most animals that have X and Y sex chromosomes, chromosome-wide mechanisms are used to balance X-linked gene expression in males and females. In the fly Drosophila melanogaster, the dosage compensation mechanism also generally extends to X-linked transgenes. Over 70 transgenic lines of the Australian sheep blowfly Lucilia cuprina have been made...
Citations
... The Drosophila gene Painting-of-fourth (POF) has been shown to mediate dosage compensation 25 in the sheep blowfly, Lucilia cuprina, a dipteran species with the ancestral element F as the X 26 (Linger et al. 2015). Given the homology between the P. cognata and the ancestral dipteran X 27 chromosome, we investigated whether POF showed patterns of expression consistent with a role 28 in dosage compensation in scorpionflies, i.e. whether it was expressed primarily in male somatic 29 ...
Many insects carry an ancient X chromosome - the Drosophila Muller element F - that likely predates their origin. Interestingly, the X has undergone turnover in multiple fly species (Diptera) after being conserved for more than 450 MY. The long evolutionary distance between Diptera and other sequenced insect clades makes it difficult to infer what could have contributed to this sudden increase in rate of turnover. Here, we produce the first genome and transcriptome of a long overlooked sister-order to Diptera: Mecoptera. We compare the scorpionfly Panorpa cognata X-chromosome gene content, expression, and structure, to that of several dipteran species as well as more distantly-related insect orders (Orthoptera and Blattodea). We find high conservation of gene content between the mecopteran X and the dipteran Muller F element, as well as several shared biological features, such as the presence of dosage compensation and a low amount of genetic diversity, consistent with a low recombination rate. However, the two homologous X chromosomes differ strikingly in their size and number of genes they carry. Our results therefore support a common ancestry of the mecopteran and ancestral dipteran X chromosomes, and suggest that Muller element F shrank in size and gene content after the split of Diptera and Mecoptera, which may have contributed to its turnover in dipteran insects.
... For CQ analysis, genomic DNA was prepared from adult males and female flies as previously described. 17 Illumina DNA library preparation and Illumina DNA sequencing were performed by the Genomic Sciences Laboratory at North Carolina State University following standard procedures. ...
... The dot chromosome of D. melanogaster was previously shown to bean ancient X chromosome in Diptera.40 In the related Australian sheep blowfly Lucilia cuprina, most of the X-linked genes are orthologs of the dot chromosome genes.17,41 Thus, it is likely that scaffold 315 corresponds to the euchromatic part of the X chromosome. ...
The New World Screwworm, Cochliomyia hominivorax (Calliphoridae), is the most important myiasis-causing species in America. Screwworm myiasis is a zoonosis that can cause severe lesions in livestock, domesticated and wild animals, and occasionally in people. Beyond the sanitary problems associated with this species, these infestations negatively impact economic sectors, such as the cattle industry.
Here, we present a chromosome-scale assembly of C. hominivorax's genome, organized in 6 chromosome-length and 515 unplaced scaffolds and spanning 534 Mb. There was a clear correspondence between the D. melanogaster linkage groups A-E and the chromosomal-scale scaffolds. Chromosome Quotient (CQ) analysis identified a single scaffold from the X chromosome that contains most of the orthologs of genes that are on the D. melanogaster fourth chromosome (linkage group F or dot chromosome). CQ analysis also identified potential X and Y unplaced scaffolds and genes. Y-linkage for selected regions was confirmed by PCR with male and female DNA. Some of the long chromosome-scale scaffolds include Y-linked sequences, suggesting misassembly of these regions. These resources will provide a basis for future studies aiming at understanding the biology and evolution of this devastating obligate parasite.
... Most of the genes that are on the X chromosome in L. cuprina map to the tiny fourth or dot chromosome in D. melanogaster [17]. X-linked genes are dosage compensated in L. cuprina [17,18]. The L. cuprina no blokes (nbl) gene is essential for male viability and normal X chromosome gene expression [17]. ...
... The mildest phenotype was otherwise normal females with bent ovipositors. The X-linked LctraIR12X line produced offspring with the least masculinization, which was expected as the large X chromosome is mostly heterochromatic and X-linked transgenes typically show lower levels of expression [18,26] (Fig 1D). No transformation was observed among the offspring of crosses with the DR2 line ( Fig 1C). ...
... The X:A ratio was higher in wildtype XX than wildtype XY ( Fig 5C) as the RNA levels for the 54 X-linked genes are on average slightly higher in females than males (average female: male ratio of 1.12). We next verified these results by qRT-PCR analysis of 5 selected X-linked genes, previously shown to be dosage compensated [17,18]. The normalized RNA levels for four of the five genes were significantly higher in XX DR3#6; LctraIR7 pupae compared to XY pupae (S3A Fig). ...
The transformer ( tra ) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina . Sex-specific tra RNA splicing is controlled by Sex lethal ( Sxl ) in Drosophila melanogaster but is auto-regulated in L . cuprina . Sxl also represses X chromosome dosage compensation in female D . melanogaster . We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L . cuprina . In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L . cuprina . However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos.
... High molecular weight DNA was isolated from mixed sex late-stage embryos of the inbred strain Ls3-8 SP2 using procedures described previously [68]. The size of the DNA was estimated by using agarose gel electrophoresis with a pippin pulse power supply (Sage Science) following conditions recommended by the manufacturer. ...
... Total RNA was extracted from frozen samples using Trizol as described previously [68]. Purification of mRNA and cDNA library preparation for Illumina DNA sequencing was as previously described [68]. ...
... Total RNA was extracted from frozen samples using Trizol as described previously [68]. Purification of mRNA and cDNA library preparation for Illumina DNA sequencing was as previously described [68]. The final quantified libraries were pooled in equimolar amounts for clustering and sequencing on an Illumina NextSeq 500 DNA sequencer. ...
The common green bottle blow fly Lucilia sericata (family, Calliphoridae) is widely used for maggot debridement therapy, which involves the application of sterile maggots to wounds. The larval excretions and secretions are important for consuming necrotic tissue and inhibiting bacterial growth in wounds of patients. Linothele sericata is also of importance as a pest of sheep and in forensic studies to estimate a postmortem interval. Here we report the assembly of a 565.3 Mb genome from long read PacBio DNA sequencing of genomic DNA. The genome contains 14,704 predicted protein coding genes and 1709 non-coding genes. Targeted annotation and transcriptional analyses identified genes that are highly expressed in the larval salivary glands (secretions) and Malpighian tubules (excretions) under normal growth conditions and following heat stress. The genomic resources will underpin future genetic studies and in development of engineered strains for genetic control of L. sericata and for biotechnology-enhanced maggot therapy.
... Summary of the current knowledge on presence/absence of dosage balance across animals. Animal species are split to groups reflecting putative independent origins of sex chromosomes (see [29][30][31][32] for evidence on homology of sex chromosomes in dipteran insects). Most evidence was taken from the review by Gu & Walters [6], supplemented by newer data (references by the individual species in the table The Y chromosomes in both species of the genus Lialis contain extensive heterochromatic blocks and accumulations of repetitive motifs, indicating a high degree of degeneration of the Y chromosome [40]. ...
Differentiation of sex chromosomes is thought to have evolved with cessation of recombination and subsequent loss of genes from the degenerated partner (Y and W) of sex chromosomes, which in turn leads to imbalance of gene dosage between sexes. Based on work with traditional model species, theory suggests that unequal gene copy numbers lead to the evolution of mechanisms to counter this imbalance. Dosage compensation, or at least achieving dosage balance in expression of sex-linked genes between sexes, has largely been documented in lineages with male heterogamety (XX/XY sex determination), while ZZ/ZW systems are assumed to be usually associated with the lack of chromosome-wide gene dose regulatory mechanisms. Here, we document that although the pygopodid geckos evolved male heterogamety with a degenerated Y chromosome 32–72 Ma, one species in particular, Burton's legless lizard ( Lialis burtonis ), does not possess dosage balance in the expression of genes in its X-specific region. We summarize studies on gene dose regulatory mechanisms in animals and conclude that there is in them no significant dichotomy between male and female heterogamety. We speculate that gene dose regulatory mechanisms are likely to be related to the general mechanisms of sex determination instead of type of heterogamety.
This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.
... The pseudo-chromosomes of A. grahami and the corresponding Muller elements of D. melanogaster are listed (Table S11). The Muller F was reported as the X-chromosome linked in some calyptratae species [98,99]. In the present study, however, it is hard to tell from the results Figure 5: Hi-C interaction matrix maps within and among 6 chromosomes. ...
Background
Blowflies (Diptera: Calliphoridae) are the most commonly found entomological evidence in forensic investigations. Distinguished from other blowflies, Aldrichina grahami has some unique biological characteristics and is a species of forensic importance. Its development rate, pattern, and life cycle can provide valuable information for the estimation of the minimum postmortem interval.
Findings
Herein we provide a chromosome-level genome assembly of A. grahami that was generated by Pacific BioSciences sequencing platform and chromosome conformation capture (Hi-C) technology. A total of 50.15 Gb clean reads of the A. grahami genome were generated. FALCON and Wtdbg were used to construct the genome of A. grahami, resulting in an assembly of 600 Mb and 1,604 contigs with an N50 size of 1.93 Mb. We predicted 12,823 protein-coding genes, 99.8% of which was functionally annotated on the basis of the de novo genome (SRA: PRJNA513084) and transcriptome (SRA: SRX5207346) of A. grahami. According to the co-analysis with 11 other insect species, clustering and phylogenetic reconstruction of gene families were performed. Using Hi-C sequencing, a chromosome-level assembly of 6 chromosomes was generated with scaffold N50 of 104.7 Mb. Of these scaffolds, 96.4% were anchored to the total A. grahami genome contig bases.
Conclusions
The present study provides a robust genome reference for A. grahami that supplements vital genetic information for nonhuman forensic genomics and facilitates the future research of A. grahami and other necrophagous blowfly species used in forensic medicine.
... C t values were called and averaged for each triplicate by SDS software (version 2.4). Samples were normalized to LcGST1 (Bagnall and Kotze 2010) and relative expression levels were calculated using the formula 2 −∆∆Ct as described previously (Linger et al. 2015). Graphs were generated using Microsoft Excel. ...
For genetic approaches for controlling insect pests such as the sterile insect technique (SIT), it is advantageous to release only males as females are ineffective as control agents and they consume about 50% of the diet. Here we developed tetracycline-repressible Lucilia cuprina transgenic strains in which adult females were fully fertile and viable on a diet that lacked tetracycline and all of their female offspring died at the embryo stage. The transgenic strains are an improvement over the strains we developed previously, which had the disadvantage that adult females on diet without tetracycline were sterile and died prematurely. This was possibly due to the low level expression of the effector gene in ovaries. In the strains developed in this study, the early promoters from L. cuprina nullo or Cochliomyia macellaria CG14427 genes were used to drive the tetracycline transactivator (tTA) expression in the early embryo. In the absence of tetracycline, tTA activates expression of the proapoptotic gene Lshid which contains a female-specific intron. Consequently, only females produce active HID protein and die at the embryo stage. Crossing the tTA-expressing driver lines with an RFPex reporter line confirmed that there was no expression of the effector gene in the ovary. These new embryonic L. cuprina transgenic sexing strains hold great promise for genetic control programs and the system reported here might also be transferable to other major calliphorid livestock pests such as the New World screwworm, Cochliomyia hominivorax.
... In addition, the Y 49 chromosomes of L. cuprina and S. bullata are extremely differenti-50 ated from their homologous X chromosomes, suggesting that they 51 have existed as an X-Y pair for many millions of years (Vicoso and 52 Bachtrog 2013Bachtrog , 2015. Furthermore, the haploid X chromosome in 53 L. cuprina males is up-regulated (i.e., dosage compensated) by an 54 RNA-binding protein that is homologous to a Drosophila protein 55 that localizes nearly exclusively to element F ( Linger et al. 2015;56 Davis et al. 2018). As expected because of the genetic differentia-57 tion between the L. cuprina X and Y, loss of function mutations in 58 the L. cuprina gene encoding the dosage compensation protein are 59 lethal specifically in males (Davis et al. 2018). ...
... Each scenario makes specific predictions about 54 the male-determining genes and sex chromosomes in the MRCA 55 of Muscidae and in other muscid species. Notably, the scenarios 56 include two important factors that could allow for faster rates 57 of sex chromosome evolution in some taxa (e.g., Muscidae) than (Vicoso and Bachtrog 2013;Linger et al. 2015;62 Vicoso and Bachtrog 2015;Davis et al. 2018). Undifferentiated sex 63 chromosomes could allow for the formation of new sex chromo-64 somes ( Stöck et al. 2011Stöck et al. , 2013Dufresnes et al. 2015). ...
Sex chromosomes and sex determining genes can evolve fast, with the sex-linked chromosomes often differing between closely related species. Population genetics theory has been developed and tested to explain the rapid evolution of sex chromosomes and sex determination. However, we do not know why the sex chromosomes are divergent in some taxa and conserved in others. Addressing this question requires comparing closely related taxa with conserved and divergent sex chromosomes to identify biological features that could explain these differences. Cytological karyotypes suggest that muscid flies (e.g., house fly) and blow flies are such a taxonomic pair. The sex chromosomes appear to differ across muscid species, whereas they are conserved across blow flies. Despite the cytological evidence, we do not know the extent to which muscid sex chromosomes are independently derived along different evolutionary lineages. To address that question, we used genomic and transcriptomic sequence data to identify young sex chromosomes in two closely related muscid species, horn fly (Haematobia irritans) and stable fly (Stomoxys calcitrans). We provide evidence that the nascent sex chromosomes of horn fly and stable fly were derived independently from each other and from the young sex chromosomes of the closely related house fly (Musca domestica). We present three different scenarios that could have given rise to the sex chromosomes of horn fly and stable fly, and we describe how the scenarios could be distinguished. Distinguishing between these scenarios in future work could identify features of muscid genomes that promote sex chromosome divergence.
... In D. melanogaster, where element F is autosomal, Painting of fourth (Pof ) encodes an RNA-binding protein that localizes predominantly to element F [41]. Lucilia cuprina (Australian sheep blowfly) has the ancestral brachyceran karyotype, with an X-linked element F [42,43]. Expression of X-linked genes is upregulated in L. cuprina males by the homolog of Pof [42,44]. ...
... Lucilia cuprina (Australian sheep blowfly) has the ancestral brachyceran karyotype, with an X-linked element F [42,43]. Expression of X-linked genes is upregulated in L. cuprina males by the homolog of Pof [42,44]. This dosage compensation is essential for male viability-a loss of function mutation in the L. cuprina homolog of Pof is male lethal, but viable in females [44]. ...
... In some fly species where element F is the X chromosome, X-linked genes are present in a single (haploid) copy in males [18]. Males of the blow fly L. cuprina are haploid for such an X chromosome, and their X-linked genes are upregulated by an RNA-binding protein encoded by a homolog of Drosophila Pof [42,44]. POF localizes nearly exclusively to element F gene bodies in D. melanogaster [41,[50][51][52]. ...
Background:
Sex chromosome evolution is a dynamic process that can proceed at varying rates across lineages. For example, different chromosomes can be sex-linked between closely related species, whereas other sex chromosomes have been conserved for > 100 million years. Cases of long-term sex chromosome conservation could be informative of factors that constrain sex chromosome evolution. Cytological similarities between the X chromosomes of the German cockroach (Blattella germanica) and most flies suggest that they may be homologous-possibly representing an extreme case of long-term conservation.
Results:
To test the hypothesis that the cockroach and fly X chromosomes are homologous, we analyzed whole-genome sequence data from cockroaches. We found evidence in both sequencing coverage and heterozygosity that a significant excess of the same genes are on both the cockroach and fly X chromosomes. We also present evidence that the candidate X-linked cockroach genes may be dosage compensated in hemizygous males. Consistent with this hypothesis, three regulators of transcription and chromatin on the fly X chromosome are conserved in the cockroach genome.
Conclusions:
Our results support our hypothesis that the German cockroach shares the same X chromosome as most flies. This may represent the convergent evolution of the X chromosome in the lineages leading to cockroaches and flies. Alternatively, the common ancestor of most insects may have had an X chromosome that resembled the extant cockroach and fly X. Cockroaches and flies diverged ∼ 400 million years ago, which would be the longest documented conservation of a sex chromosome. Cockroaches and flies have different mechanisms of sex determination, raising the possibility that the X chromosome was conserved despite the evolution of the sex determination pathway.
... In D. melanogaster, where element F is autosomal, Painting of fourth (Pof ) encodes an RNA-binding protein that localizes predominantly to element F [40]. Lucilia cuprina (Australian sheep blowfly) has the ancestral brachyceran karyotype, with an X-linked element F [41,42]. Expression of X-linked genes is up-regulated in L. cuprina males by the homolog of Pof [41,43]. ...
... Lucilia cuprina (Australian sheep blowfly) has the ancestral brachyceran karyotype, with an X-linked element F [41,42]. Expression of X-linked genes is up-regulated in L. cuprina males by the homolog of Pof [41,43]. This dosage compensation is essential for male viability-a loss of function mutation in the L. cuprina homolog of Pof is male lethal, but viable in females [43]. ...
... In some fly species where element F is the X chromosome, X-linked genes are present in a single (haploid) copy in males [27]. Males of the blow fly L. cuprina are haploid for such an X chromosome, and their X-linked genes are up-regulated by an RNAbinding protein encoded by a homolog of Drosophila Pof [41,43]. POF localizes nearly exclusively to element F gene bodies in D. melanogaster [40,[49][50][51]. ...
Background
Sex chromosome evolution is a dynamic process that can proceed at varying rates across lineages. For example, different chromosomes can be sex-linked between closely related species, whereas other sex chromosomes have been conserved for > 100 million years. Cases of long-term sex chromosome conservation could be informative of factors that constrain sex chromosome evolution. Cytological similarities between the X chromosomes of the German cockroach ( Blattella germanica ) and most flies suggest that they may be homologous—possibly representing an extreme case of long-term conservation.
Results
To test the hypothesis that the cockroach and fly X chromosomes are homologous, we analyzed whole genome sequence data from cockroach. We found evidence in both sequencing coverage and heterozygosity that a significant excess of the same genes are on both the cockroach and fly X chromosomes. We also present evidence that the candidate X-linked cockroach genes may be dosage compensated in hemizygous males. Consistent with this hypothesis, three regulators of transcription and chromatin on the fly X chromosome are conserved in the cockroach genome.
Conclusions
Our results support our hypothesis that the German cockroach shares the same X chromosome as most flies. This may represent convergent evolution of the X chromosome in the lineages leading to cockroaches and flies. Alternatively, the common ancestor of most insects may have had an X chromosome that resembled the extant cockroach and fly X. Cockroaches and flies diverged ∼400 million years ago, which would be the longest documented conservation of a sex chromosome. Cockroaches and flies have different mechanisms of sex determination, raising the possibility that the X chromosome was conserved despite evolution of the sex determination pathway.
