BackgroundThe New World screwworm, Cochliomyia hominivorax, is a devastating pest of livestock endemic to subtropical and tropical regions of the Western hemisphere. The larvae of this species feed on the tissue of living animals, including man, and can cause death if untreated. Over 60 years ago, the sterile insect technique (SIT) was developed with the aim of eradicating this pest, initially from Florida but subsequently from all of North and Central America. From the outset it was appreciated that SIT would be more efficient if only sterile males were released in the field, but this was not possible until now.ResultsHere, we report on the development and evaluation of the first sexing strains of C. hominivorax that produce only males when raised on diet without tetracycline. Transgenic lines have been developed that possess a tetracycline repressible female-lethal genetic system. Ten of these lines show high female lethality at the late larval/pupal stages and three of them present dominant female lethality. Most of the lines were comparable to the wild type parental strain in several fitness parameters that are relevant to mass rearing in a production facility. Further, three lines performed well in male mating success and male competition assays, suggesting they would be sexually competitive in the field. Consequently, one transgenic line has been selected by the New World Screwworm Program for evaluation under mass rearing conditions.ConclusionsWe conclude that the promising characteristics of the selected sexing strains may contribute to reduce production costs for the existing eradication program and provide more efficient population suppression, which should make a genetic control program more economical in regions were C. hominivorax remains endemic.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0296-8) contains supplementary material, which is available to authorized users.

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.

The New World Screwworm fly, Cochliomyia hominivorax, is a major pest of livestock in South America and Caribbean. However, few genomic resources have been available for this species. A genome of 534 Mb was assembled from long read PacBio DNA sequencing of DNA from a highly inbred strain. Analysis of molecular evolution identified 40 genes that are likely under positive selection. Developmental RNA-seq analysis identified specific genes associated with each stage. We identify and analyze the expression of genes that are likely important for host-seeking behavior (chemosensory), development of larvae in open wounds in warm-blooded animals (heat shock protein, immune response) and for building transgenic strains for genetic control programs including gene drive (sex determination, germline). This study will underpin future experiments aimed at understanding the parasitic lifestyle of the screwworm fly and greatly facilitate future development of strains for efficient systems for genetic control of screwworm.

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o objetivo de fornecer insetos para programas para sua erradicação, através da técnica do inseto estéril. Novos métodos de criação têm auxiliado na redução dos custos desses programas de erradicação. Nós examinamos o efeito e a interação de três temperaturas (24,5, 29,5 e 34,5 o C), duas dietas (carboidrato e carboidrato + proteína) e três densidades populacionais (300, 400 e 500 moscas/gaiola), na fecundidade eclosão de larvas, número de ovos fertilizados, mortalidade (macho e fêmea) e desenvolvimento dos ovários. As três densidades populacionais não afetaram nenhum dos parâmetros avaliados. O uso da dieta protéica aumentou a produção de ovos em todas as temperaturas testadas. O tipo de dieta não afetou a eclosão de larvas e mortalidade de fêmeas. A mortalidade dos machos foi signifi cativamente maior quando alimentados com a dieta protéica e criados a 24,5o C e 34,5 o C. A eclosão de larvas foi signifi cativamente menor quando as moscas foram criadas a 34,5 o C. Nas temperaturas mais elevadas (37 o C e 40 o C), a produção de ovos, eclosão de larvas, fertilidade e mortalidade foram afetadas desfavoravelmente. Ainda nessas mesmas temperaturas, durante a oogênese, não houve a formação adequada do estoque de alimento para os embriões. O fotoperíodo curto (1L: 23E) propiciou maior produção de ovos, eclosão de larvas e fertilidade, e diminuiu a mortalidade quando comparado ao fotoperíodo padrão em criações (12L: 12E). PALAVRAS-CHAVE: Temperatura, produção de ovos, desenvolvimento do ovário ABSTRACT -The New World screwworm, Cochliomyia hominivorax Coquerel, is mass reared for screwworm eradication initiatives that use the sterile insect technique. New methods for rearing have helped to reduce the cost of the eradication program. We examined the effect and interaction of three temperatures (24.5, 29.5 and 34.5ºC), two diets (2% spray-dried blood plus 0.05% vitamins and corn syrup carrageenan) and three population densities (300, 400, and 500 fl ies/cage) on egg production, egg hatch, number of observable fertilized eggs, mortality (male and female) and ovarian development. The three population densities did not affect any of the parameters monitored. Using the protein diet increased egg production at all temperatures. Diet did not affect egg hatch or female mortality. Male mortality was signifi cantly greater when fed the protein diet and reared at 24.5ºC and 34.5ºC. Egg hatch was signifi cantly less when the fl ies were reared at 34.5ºC. When exposed to high temperatures (37ºC and 40ºC) egg production, egg hatch, fertility and mortality were adversely affected. At the higher temperatures, yolk did not adequately form during oogenesis. When compared to the normal rearing photoperiod (12 L: 12 D), short photoperiod (1 L: 23 D) increased egg production, egg hatch and fertility but lowered mortality.