@article{li_yamamoto_belikoff_berger_griffith_scott_2021, title={A conditional female lethal system for genetic suppression of the global fruit crop pest Drosophila suzukii}, ISSN={["1526-4998"]}, DOI={10.1002/ps.6530}, abstractNote={Abstract BACKGROUND Drosophila suzukii (Matsumura, 1931, Diptera: Drosophilidae) is a global pest of soft‐skinned fruits such as blueberries, cherries and raspberries. Also known as spotted‐wing drosophila, D. suzukii is native to Asia but is now widely distributed in the Americas and Europe, and presents a serious challenge for growers. Genetic control strategies offer an environmentally friendly approach for the control of D. suzukii . RESULTS In this study, we developed transgenic strains of D. suzukii that carry dominant conditional female lethal transgenes. When raised in the absence of tetracycline, female D. suzukii die. We show that repeated releases of an excess of transgenic males can suppress D. suzukii populations in laboratory cage trials. CONCLUSION Our data suggest that the transgenic strain could provide an effective approach for control of this invasive pest of soft‐skinned fruits.}, journal={PEST MANAGEMENT SCIENCE}, author={Li, Fang and Yamamoto, Akihiko and Belikoff, Esther J. and Berger, Amy and Griffith, Emily H. and Scott, Maxwell J.}, year={2021}, month={Jul} }
 @article{davis_belikoff_dickey_scholl_benoit_scott_2021, title={Genome and transcriptome sequencing of the green bottle fly, Lucilia sericata, reveals underlying factors of sheep flystrike and maggot debridement therapy}, volume={113}, ISSN={["1089-8646"]}, DOI={10.1016/j.ygeno.2021.10.003}, abstractNote={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. Lucilia 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.}, number={6}, journal={GENOMICS}, author={Davis, Rebecca J. and Belikoff, Esther J. and Dickey, Allison N. and Scholl, Elizabeth H. and Benoit, Joshua B. and Scott, Maxwell J.}, year={2021}, month={Nov}, pages={3978–3988} }
 @article{paulo_williamson_arp_li_sagel_skoda_sanchez-gallego_vasquez_quintero_leon_et al._2019, title={Specific Gene Disruption in the Major Livestock Pests Cochliomyia hominivorax and Lucilia cuprina Using CRISPR/Cas9}, volume={9}, ISSN={["2160-1836"]}, DOI={10.1534/g3.119.400544}, abstractNote={Abstract Cochliomyia hominivorax and Lucilia cuprina are major pests of livestock. Their larvae infest warm-blooded vertebrates and feed on host’s tissues, resulting in severe industry losses. As they are serious pests, considerable effort has been made to develop genomic resources and functional tools aiming to improve their management and control. Here, we report a significant addition to the pool of genome manipulation tools through the establishment of efficient CRISPR/Cas9 protocols for the generation of directed and inheritable modifications in the genome of these flies. Site-directed mutations were introduced in the C. hominivorax and L. cuprina yellow genes (ChY and LcY) producing lightly pigmented adults. High rates of somatic mosaicism were induced when embryos were injected with Cas9 ribonucleoprotein complexes (RNPs) pre-assembled with guide RNAs (sgRNAs) at high concentrations. Adult flies carrying disrupted yellow alleles lacked normal pigmentation (brown body phenotype) and efficiently transmitted the mutated alleles to the subsequent generation, allowing the rapid creation of homozygous strains for reverse genetics of candidate loci. We next used our established CRISPR protocol to disrupt the C. hominivorax transformer gene (Chtra). Surviving females carrying mutations in the Chtra locus developed mosaic phenotypes of transformed ovipositors with characteristics of male genitalia while exhibiting abnormal reproductive tissues. The CRISPR protocol described here is a significant improvement on the existing toolkit of molecular methods in calliphorids. Our results also suggest that Cas9-based systems targeting Chtra and Lctra could be an effective means for controlling natural populations of these important pests.}, number={9}, journal={G3-GENES GENOMES GENETICS}, author={Paulo, Daniel F. and Williamson, Megan E. and Arp, Alex P. and Li, Fang and Sagel, Agustin and Skoda, Steven R. and Sanchez-Gallego, Joel and Vasquez, Mario and Quintero, Gladys and Leon, Adalberto A. and et al.}, year={2019}, month={Sep}, pages={3045–3055} }
 @article{davis_belikoff_scott_2018, title={Towards next generation maggot debridement therapy: Transgenic Lucilia sericata larvae that produce and secrete a human growth factor}, volume={26}, number={1}, journal={Wound Repair and Regeneration}, author={Davis, R. J. and Belikoff, E. J. and Scott, M. J.}, year={2018}, pages={A27–27} }
 @article{davis_belikoff_scholl_li_scott_2018, title={no blokes Is Essential for Male Viability and X Chromosome Gene Expression in the Australian Sheep Blowfly}, volume={28}, ISSN={["1879-0445"]}, DOI={10.1016/j.cub.2018.05.005}, abstractNote={It has been hypothesized that the Drosophila 4th chromosome is derived from an ancient X chromosome [1Vicoso B. Bachtrog D. Reversal of an ancient sex chromosome to an autosome in Drosophila.Nature. 2013; 499: 332-335Crossref PubMed Scopus (131) Google Scholar]. In the Australian sheep blowfly, Lucilia cuprina, the heterochromatic X chromosome contains few active genes and orthologs of Drosophila X-linked genes are autosomal. Of 8 X-linked genes identified previously in L. cuprina, 6 were orthologs of Drosophila 4th-chromosome genes [2Linger R.J. Belikoff E.J. Scott M.J. Dosage compensation of X-linked Muller element F genes but not X-linked transgenes in the Australian sheep blowfly.PLoS ONE. 2015; 10: e0141544Crossref PubMed Scopus (14) Google Scholar]. The X-linked genes were expressed equally in males and females. Here we identify an additional 51 X-linked genes and show that most are dosage compensated. Orthologs of 49 of the 59 X-linked genes are on the 4th chromosome in D. melanogaster. Because painting of fourth (Pof) is important for expression of Drosophila 4th-chromosome genes [3Johansson A.M. Stenberg P. Allgardsson A. Larsson J. POF regulates the expression of genes on the fourth chromosome in Drosophila melanogaster by binding to nascent RNA.Mol. Cell. Biol. 2012; 32: 2121-2134Crossref PubMed Scopus (27) Google Scholar], we used Cas9 to make a loss-of-function knockin mutation in an L. cuprina Pof ortholog we call no blokes (nbl). Homozygous nbl males derived from homozygous nbl mothers die at the late pupal stage. Homozygous nbl females are viable, fertile, and live longer than heterozygous nbl females. RNA expression of most X-linked genes was reduced in homozygous nbl male pupae and to a lesser extent in nbl females compared to heterozygous siblings. The results suggest that NBL could be important for X chromosome dosage compensation in L. cuprina. NBL may also facilitate gene expression in the heterochromatic environment of the X chromosome in both sexes. This study supports the hypothesis on the origin of the Drosophila 4th chromosome and that a POF-like protein was required for normal gene expression on the ancient X chromosome.}, number={12}, journal={CURRENT BIOLOGY}, author={Davis, Rebecca J. and Belikoff, Esther J. and Scholl, Elizabeth H. and Li, Fang and Scott, Maxwell J.}, year={2018}, month={Jun}, pages={1987-+} }
 @article{linger_belikoff_yan_li_wantuch_fitzsimons_scott_2016, title={Towards next generation maggot debridement therapy: transgenic Lucilia sericata larvae that produce and secrete a human growth factor}, volume={16}, ISSN={["1472-6750"]}, DOI={10.1186/s12896-016-0263-z}, abstractNote={Diabetes and its concurrent complications impact a significant proportion of the population of the US and create a large financial burden on the American health care system. FDA-approved maggot debridement therapy (MDT), the application of sterile laboratory-reared Lucilia sericata (green bottle fly) larvae to wounds, is a cost-effective and successful treatment for diabetic foot ulcers and other medical conditions. Human platelet derived growth factor-BB (PDGF-BB) is a secreted dimeric peptide growth factor that binds the PDGF receptor. PDGF-BB stimulates cell proliferation and survival, promotes wound healing, and has been investigated as a possible topical treatment for non-healing wounds. Genetic engineering has allowed for expression and secretion of human growth factors and other proteins in transgenic insects. Here, we present a novel concept in MDT technology that combines the established benefits of MDT with the power of genetic engineering to promote healing. The focus of this study is to create and characterize strains of transgenic L. sericata that express and secrete PDGF-BB at detectable levels in adult hemolymph, whole larval lysate, and maggot excretions/ secretions (ES), with potential for clinical utility in wound healing. We have engineered and confirmed transgene insertion in several strains of L. sericata that express human PDGF-BB. Using a heat-inducible promoter to control the pdgf-b gene, pdgf-b mRNA was detected via semi-quantitative PCR upon heat shock. PDGF-BB protein was also detectable in larval lysates and adult hemolymph but not larval ES. An alternative, tetracycline-repressible pdgf-b system mediated expression of pdgf-b mRNA when maggots were raised on diet that lacked tetracycline. Further, PDGF-BB protein was readily detected in whole larval lysate as well as larval ES. Here we show robust, inducible expression and production of human PDGF-BB protein from two conditional expression systems in transgenic L. sericata larvae. The tetracycline-repressible system appears to be the most promising as PDGF-BB protein was detectable in larval ES following induction. Our system could potentially be used to deliver a variety of growth factors and anti-microbial peptides to the wound environment with the aim of enhancing wound healing, thereby improving patient outcome in a cost-effective manner.}, journal={BMC BIOTECHNOLOGY}, author={Linger, Rebecca J. and Belikoff, Esther J. and Yan, Ying and Li, Fang and Wantuch, Holly A. and Fitzsimons, Helen L. and Scott, Maxwell J.}, year={2016}, month={Mar} }
 @article{linger_belikoff_scott_2015, title={Dosage Compensation of X-Linked Muller Element F Genes but Not X-Linked Transgenes in the Australian Sheep Blowfly}, volume={10}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0141544}, abstractNote={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 as part of an effort to develop male-only strains for a genetic control program of this major pest of sheep. All lines carry a constitutively expressed fluorescent protein marker gene. In all 12 X-linked lines, female larvae show brighter fluorescence than male larvae, suggesting the marker gene is not dosage compensated. This has been confirmed by quantitative RT-PCR for selected lines. To determine if endogenous X-linked genes are dosage compensated, we isolated 8 genes that are orthologs of genes that are on the fourth chromosome in D. melanogaster. Recent evidence suggests that the D. melanogaster fourth chromosome, or Muller element F, is the ancestral X chromosome in Diptera that has reverted to an autosome in Drosophila species. We show by quantitative PCR of male and female DNA that 6 of the 8 linkage group F genes reside on the X chromosome in L. cuprina. The other two Muller element F genes were found to be autosomal in L. cuprina, whereas two Muller element B genes were found on the same region of the X chromosome as the L. cuprina orthologs of the D. melanogaster Ephrin and gawky genes. We find that the L. cuprina X chromosome genes are equally expressed in males and females (i.e., fully dosage compensated). Thus, unlike in Drosophila, it appears that the Lucilia dosage compensation system is specific for genes endogenous to the X chromosome and cannot be co-opted by recently arrived transgenes.}, number={10}, journal={PLOS ONE}, author={Linger, Rebecca J. and Belikoff, Esther J. and Scott, Maxwell J.}, year={2015}, month={Oct} }
 @article{edman_linger_belikoff_li_sze_tarone_scott_2015, title={Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos}, volume={24}, ISSN={["1365-2583"]}, DOI={10.1111/imb.12135}, abstractNote={The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area-wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making ‘male-only’ strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro-apoptotic gene by the tetracycline-dependent transactivator. Sex-specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro-apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro-apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro-apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests.}, number={1}, journal={INSECT MOLECULAR BIOLOGY}, author={Edman, R. M. and Linger, R. J. and Belikoff, E. J. and Li, F. and Sze, S. -H. and Tarone, A. M. and Scott, M. J.}, year={2015}, month={Feb}, pages={58–70} }
 @article{li_wantuch_linger_belikoff_scott_2014, title={Transgenic sexing system for genetic control of the Australian sheep blow fly Lucilia cuprina}, volume={51}, ISSN={["1879-0240"]}, DOI={10.1016/j.ibmb.2014.06.001}, abstractNote={The New World screwworm and the Australian sheep blowfly Lucilia cuprina are devastating pests of livestock. The larvae of these species feed on the tissue of the living animal and can cause death if untreated. The sterile insect technique or SIT was used to eradicate screwworm from North and Central America. This inspired efforts to develop strains containing complex chromosomal rearrangements for genetic control of L. cuprina in Australia. Although one field trial was promising, the approach was abandoned due to costs and difficulties in mass rearing the strain. As the efficiency of SIT can be significantly increased if only sterile males are released, we have developed transgenic strains of L. cuprina that carry a dominant tetracycline repressible female lethal genetic system. Lethality is due to overexpression of an auto-regulated tetracycline repressible transactivator (tTA) gene and occurs mostly at the pupal stage. Dominant female lethality was achieved by replacing the Drosophila hsp70 core promoter with a Lucilia hsp70 core promoter-5′UTR for tTA overexpression. The strains carry a dominant strongly expressed marker that will facilitate identification in the field. Interestingly, the sexes could be reliably sorted by fluorescence or color from the early first instar larval stage as females that overexpress tTA also overexpress the linked marker gene. Male-only strains of L. cuprina developed in this study could form the basis for a future genetic control program. Moreover, the system developed for L. cuprina should be readily transferrable to other major calliphorid livestock pests including the New and Old World screwworm.}, journal={INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY}, author={Li, Fang and Wantuch, Holly A. and Linger, Rebecca J. and Belikoff, Esther J. and Scott, Maxwell J.}, year={2014}, month={Aug}, pages={80–88} }
 @article{li_vensko_belikoff_scott_2013, title={Conservation and Sex-Specific Splicing of the transformer Gene in the Calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata}, volume={8}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0056303}, abstractNote={Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3' end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a "male-only" strain for genetic control programs.}, number={2}, journal={PLOS ONE}, author={Li, Fang and Vensko, Steven P., II and Belikoff, Esther J. and Scott, Maxwell J.}, year={2013}, month={Feb} }
 @article{concha_edman_belikoff_schiemann_carey_scott_2012, title={Organization and expression of the Australian sheep blowfly (Lucilia cuprina) hsp23, hsp24, hsp70 and hsp83 genes}, volume={21}, ISSN={["0962-1075"]}, DOI={10.1111/j.1365-2583.2011.01123.x}, abstractNote={In this study we report the isolation and characterization of a heat shock protein 70 (hsp70) gene, the hsp83 gene and two genes that encode small Hsps (Lchsp23 and Lchsp24) from the Australian sheep blowfly, Lucilia cuprina, a major agricultural pest. Phylogenetic analyses indicate that the LcHsp23 protein is the orthologue of Drosophila melanogaster Hsp23 and LcHsp24 is the orthologue of Sarcophaga crassipalpis Hsp23. Quantitative reverse-transcriptase PCR analysis showed that the basal level of Lchsp83 RNA is relatively high at all developmental stages and only moderately induced by heat shock. In contrast, Lchsp70 transcripts are present at low levels and strongly induced by heat shock at all stages. The basal levels of expression and degrees of heat induction of the Lchsp23 and Lchsp24 transcripts were more variable across the different developmental stages. Putative heat shock factor binding sites were identified in the Lchsp24, Lchsp70 and Lchsp83 gene promoters. The isolation of these hsp gene promoters will facilitate constitutive or conditional expression of a gene of interest in transgenic Lucilia.}, number={2}, journal={INSECT MOLECULAR BIOLOGY}, author={Concha, C. and Edman, R. M. and Belikoff, E. J. and Schiemann, A. H. and Carey, B. and Scott, M. J.}, year={2012}, month={Apr}, pages={169–180} }
 @article{schiemann_li_weake_belikoff_klemmer_moore_scott_2010, title={Sex-biased transcription enhancement by a 5 ' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila}, volume={11}, ISSN={["1471-2199"]}, DOI={10.1186/1471-2199-11-80}, abstractNote={Abstract Background In male Drosophila melanogaster , the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator. Results MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila . We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila . In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS- lacZ and UAS- arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS- arm-lacZ expression increased by about 2-fold in both sexes. Conclusions Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS- DsRed but not UAS- arm-lacZ genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.}, journal={BMC MOLECULAR BIOLOGY}, author={Schiemann, Anja H. and Li, Fang and Weake, Vikki M. and Belikoff, Esther J. and Klemmer, Kent C. and Moore, Stanley A. and Scott, Maxwell J.}, year={2010}, month={Nov} }