Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination (Lee et al., 2018). These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps) (Kanca et al., 2019a). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 50% of Drosophila genes lack suitable coding introns for integration of artificial exons. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

Sex determination is a key developmental process, yet it is remarkably variable across the tree of life. The dipteran family Sciaridae exhibits one of the most unusual sex determination systems in which mothers control offspring sex through selective elimination of paternal X chromosomes. Whereas in some members of the family females produce mixed-sex broods, others such as the dark-winged fungus gnat Bradysia coprophila are monogenic, with females producing single-sex broods. Female-producing females were previously found to be heterozygous for a large X-linked paracentric inversion (X'), which is maternally inherited and absent from male-producing females. Here we assembled and characterized the X' sequence. As close sequence homology between the X and X' made identification of the inversion challenging, we developed a k-mer-based approach to bin genomic reads before assembly. We confirmed that the inversion spans most of the X' chromosome (approximately 55Mb) and encodes around 3500 genes. Analysis of the divergence between the inversion and the homologous region of the X revealed that it originated very recently (<0.5 mya). Surprisingly, we found that the X' is more complex than previously thought and is likely to have undergone multiple rearrangements that have produced regions of varying ages, resembling a supergene composed of evolutionary strata. We found functional degradation of around 7.3% of genes within the region of recombination suppression, but no evidence of accumulation of repetitive elements. Our findings provide an indication that sex-linked inversions are driving turnover of the strange sex determination system in this family of flies.

Sex determination is a key developmental process, yet it is remarkably variable across the tree of life. The dipteran family Sciaridae exhibits one of the most unusual sex determination systems in which mothers control offspring sex through selective elimination of paternal X chromosomes. Whereas in some members of the family females produce mixed-sex broods, others such as the dark-winged fungus gnat Bradysia coprophila are monogenic, with females producing single-sex broods. Female-producing females were previously found to be heterozygous for a large X-linked paracentric inversion (X & PRIME;), which is maternally inherited and absent from male-producing females. Here, we assembled and characterized the X & PRIME; sequence. As close sequence homology between the X and X & PRIME; made identification of the inversion challenging, we developed a k-mer-based approach to bin genomic reads before assembly. We confirmed that the inversion spans most of the X & PRIME; chromosome (& SIM;55 Mb) and encodes & SIM;3,500 genes. Analysis of the divergence between the inversion and the homologous region of the X revealed that it originated very recently (<0.5 Ma). Surprisingly, we found that the X & PRIME; is more complex than previously thought and is likely to have undergone multiple rearrangements that have produced regions of varying ages, resembling a supergene composed of evolutionary strata. We found functional degradation of & SIM;7.3% of genes within the region of recombination suppression, but no evidence of accumulation of repetitive elements. Our findings provide an indication that sex-linked inversions are driving turnover of the strange sex determination system in this family of flies.

The level of resistance to radiation and the developmental and molecular responses can vary between species, and even between developmental stages of one species. For flies (order: Diptera), prior studies concluded that the fungus gnat Bradysia (Sciara) coprophila (sub-order: Nematocera) is more resistant to irradiation-induced mutations that cause visible phenotypes than the fruit fly Drosophila melanogaster (sub-order: Brachycera). Therefore, we characterized the effects of and level of resistance to ionizing radiation on B. coprophila throughout its life cycle. Our data show that B. coprophila embryos are highly sensitive to even low doses of gamma-irradiation, whereas late-stage larvae can tolerate up to 80 Gy (compared to 40 Gy for D. melanogaster) and still retain their ability to develop to adulthood, though with a developmental delay. To survey the genes involved in the early transcriptional response to irradiation of B. coprophila larvae, we compared larval RNA-seq profiles with and without radiation treatment. The up-regulated genes were enriched for DNA damage response genes, including those involved in DNA repair, cell cycle arrest, and apoptosis, whereas the down-regulated genes were enriched for developmental regulators, consistent with the developmental delay of irradiated larvae. Interestingly, members of the PARP and AGO families were highly up-regulated in the B. coprophila radiation response. We compared the transcriptome responses in B. coprophila to the transcriptome responses in D. melanogaster from 3 previous studies: whereas pathway responses are highly conserved, specific gene responses are less so. Our study lays the groundwork for future work on the radiation responses in Diptera.

The diverse cytochrome P450 enzymes of insects play essential physiological roles and also play important roles in the metabolism of environmental chemicals such as insecticides. We manually curated the complement of P450 (CYP) genes, or CYPome, of the black fungus gnat, Bradysia (Sciara) coprophila (Diptera, Sciaroidea), a species with a variable number of chromosomes. This CYPome carries two types of "alien" P450 genes. The first type of alien P450s was found among the 163 CYP genes of the core genome (autosomes and X). They consist of 28 sequences resulting from horizontal gene transfer, with closest sequences not found in insects, but in other arthropods, often Collembola. These genes are not contaminants, because they are expressed genes with introns, found in synteny with regular dipteran genes, also found in B. odoriphaga and B. hygida. Two such "alien" genes are representatives of CYP clans not otherwise found in insects, a CYP53 sequence related to fungal CYP53 genes, and a CYP19-like sequence similar to some collembolan sequences but of unclear origin. The second type of alien P450s are represented by 99 sequences from germline-restricted chromosomes (GRC). While most are P450 pseudogenes, 33 are apparently intact, with half being more closely related to P450s from Cecidomyiidae than from Sciaridae, thus supporting the hypothesis of a cross-family hybridization origin of the GRC.

One key goal of the Hubble Space Telescope Cosmic Assembly Near-Infrared Deep Extragalactic Legacy Survey is to track galaxy evolution back to z approximate to 8. Its two-tiered "wide and deep" strategy bridges significant gaps in existing near-infrared surveys. Here we report on z approximate to 8 galaxy candidates selected as F105W-band dropouts in one of its deep fields, which covers 50.1 arcmin(2) to 4 ks depth in each of three near-infrared bands in the Great Observatories Origins Deep Survey southern field. Two of our candidates have J < 26.2 mag, and are >1 mag brighter than any previously known F105W-dropouts. We derive constraints on the bright end of the rest-frame ultraviolet luminosity function of galaxies at z approximate to 8, and show that the number density of such very bright objects is higher than expected from the previous Schechter luminosity function estimates at this redshift. Another two candidates are securely detected in Spitzer Infrared Array Camera images, which are the first such individual detections at z approximate to 8. Their derived stellar masses are on the order of a few x 10(9) M-circle dot, from which we obtain the first measurement of the high-mass end of the galaxy stellar mass function at z approximate to 8. The high number density of very luminous and very massive galaxies at z approximate to 8, if real, could imply a large stellar-to-halo mass ratio and an efficient conversion of baryons to stars at such an early time.

We combine high-resolution Hubble Space Telescope/WFC3 images with multi- wavelength photometry to track the evolution of structure and activity of massive (M-* > 10(10)M(circle dot)) galaxies at redshifts z = 1.4-3 in two fields of the Cosmic Assembly Near-infrared Deep Extragalactic Legacy Survey. We detect compact, star- forming galaxies (cSFGs) whose number densities, masses, sizes, and star formation rates (SFRs) qualify them as likely progenitors of compact, quiescent, massive galaxies (cQGs) at z = 1.5-3. At z greater than or similar to 2, cSFGs present SFR = 100-200M(circle dot) yr(-1), yet their specific star formation rates (sSFR similar to 10(-9) yr(-1)) are typically half that of other massive SFGs at the same epoch, and host X-ray luminous active galactic nuclei (AGNs) 30 times (similar to 30%) more frequently. These properties suggest that cSFGs are formed by gas- rich processes (mergers or disk- instabilities) that induce a compact starburst and feed an AGN, which, in turn, quench the star formation on dynamical timescales (few 10(8) yr). The cSFGs are continuously being formed at z = 2-3 and fade to cQGs down to z similar to 1.5. After this epoch, cSFGs are rare, thereby truncating the formation of new cQGs. Meanwhile, down to z = 1, existing cQGs continue to enlarge to match local QGs in size, while less-gas-rich mergers and other secular mechanisms shepherd (larger) SFGs as later arrivals to the red sequence. In summary, we propose two evolutionary tracks of QG formation: an early (z greater than or similar to 2), formation path of rapidly quenched cSFGs fading into cQGs that later enlarge within the quiescent phase, and a late-arrival (z less than or similar to 2) path in which larger SFGs form extended QGs without passing through a compact state.

We have made a serendipitous discovery of a massive (similar to 5 x 10(11)M(circle dot)) cD galaxy at z = 1.096 in a candidate-rich cluster in the Hubble Ultra Deep Field (HUDF) area of GOODS-South. This brightest cluster galaxy (BCG) is the most distant cD galaxy confirmed to date. Ultra-deep HST/WFC3 images reveal an extended envelope starting from similar to 10 kpc and reaching similar to 70 kpc in radius along the semimajor axis. The spectral energy distributions indicate that both its inner component and outer envelope are composed of an old, passively evolving (specific star formation rate < 10-4 Gyr(-1)) stellar population. The cD galaxy lies on the same mass-size relation as the bulk of quiescent galaxies at similar redshifts. The cD galaxy has a higher stellar mass surface density (similar to M-*/R-50(2)) but a similar velocity dispersion (similar to root M-*/R-50) to those of more massive, nearby cDs. If the cD galaxy is one of the progenitors of today's more massive cDs, its size (R-50) and stellar mass have had to increase on average by factors of 3.4 +/- 1.1 and 3.3 +/- 1.3 over the past similar to 8 Gyr, respectively. Such increases in size and stellar mass without being accompanied by significant increases in velocity dispersion are consistent with evolutionary scenarios driven by both major and minor dissipationless (dry) mergers. If such cD envelopes originate from dry mergers, our discovery of even one example proves that some BCGs entered the dry merger phase at epochs earlier than z = 1. Our data match theoretical models which predict that the continuance of dry mergers at z < 1 can result in structures similar to those of massive cD galaxies seen today. Moreover, our discovery is a surprise given that the extreme depth of the HUDF is essential to reveal such an extended cD envelope at z > 1 and, yet, the HUDF covers only a minuscule region of sky (similar to 3.1 x 10(-8)). Adding that cDs are rare, our serendipitous discovery hints that such cDs may be more common than expected, perhaps even ubiquitous. Images reaching HUDF depths of more area (especially with cluster BCGs at z > 1) are needed to confirm this conjecture.