Doxycycline is a viable alternative to tetracycline for use in insect Tet-Off transgenic sexing systems, as assessed in the blowflies Cochliomyia hominivorax and Lucilia cuprina (Diptera: Calliphoridae).

Transgenic insect strains with tetracycline repressible (Tet-Off) female-lethal genes provide significant advantages over traditional sterile insect techniques for insect population control, such as reduced diet and labor costs and more efficient population suppression. Tet-Off systems are suppressed by tetracycline-class antibiotics, most commonly tetracycline (Tc) or doxycycline (Dox), allowing for equal sex ratio colonies of transgenic insects when reared with Tc or Dox and male-only generations in their absence. Dox is a more stable molecule and has increased uptake than Tc, which could be advantageous in some insect mass-rearing systems. Here, we evaluated the suitability of Dox for rearing Tet-Off female-lethal strains of Australian sheep blowfly, Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae), and New World screwworm, Cochliomyia hominivorax (Coquerel, 1858) (Diptera: Calliphoridae), and the effects of dosage on strain performance. For both species, colonies were able to be maintained with mixed-sex ratios at much lower dosages of Dox than Tc. Biological yields of C. hominivorax on either antibiotic were not significantly different. Reduction of Dox dosages in C. hominivorax diet did not affect biological performance, though rearing with 10 or 25 µg/mL was more productive than 50 µg/mL. Additionally, C. hominivorax mating performance and longevity were equal on all Dox dosages. Overall, Dox was a suitable antibiotic for mass-rearing Tet-Off female-lethal L. cuprina and C. hominivorax and was functional at much lower dosages than Tc.

An alternative chicken-based diet for mass-rearing screwworm flies.

Screwworm flies are mass-reared and released along the Panama-Colombia border to prevent reinfestation of Central and North America. The cost of the production facility, labor, and diet materials makes mass-rearing the most expensive component of the program. The mass-rearing diet has a large impact on the quality and quantity of insects produced, both of which are necessary for the successful implementation of the sterile insect technique. The diet currently used to rear screwworm flies in Panama contains dried bovine red blood cells, dried bovine plasma, egg powder, milk replacement powder, cellulose (thickening agent), formaldehyde (antimicrobial), and water. Here, we tested an alternative diet containing 2 chicken by-products, which cost less and are locally available, to replace the egg powder and milk replacement powder currently used in the diet. We used 2 screwworm colony strains in our test, the current production strain (Jamaica) and an early female-lethal strain. The chicken diet performed similarly to the production diet with the Jamaica strain, while further optimization will likely be needed for transgenic strain. Finally, nutritional analysis conducted on 7 diet ingredients will assist with diet optimization and the identification of alternative diet ingredients.

Geographic Population Genetic Structure of the New World Screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae), Using SNPs.

The New World screwworm, Cochliomyia hominivorax (Coquerel 1858) (Diptera: Calliphoridae), is a serious parasite of livestock, humans, and other warm-blooded animals. It has been eradicated from the northern parts of its historical range down to the Panama-Colombian border where a permanent barrier zone is maintained. This eradication was accomplished through using the sterile insect technique (SIT). In 2016 there was an outbreak of C. hominivorax in the Florida Keys. In only six months, this pest was successfully re-eradicated using SIT, but the geographic origin of the invasion has yet to be resolved. It was previously determined that the Florida flies most likely represented a single invasion, and it was recommended that a finer-scale genetic assessment should be completed. Thus, this current proof-of-concept study aimed to develop a population genetic database using single nucleotide polymorphisms (SNPs) to reference outbreaks and potentially identify the origin of the Florida outbreak. This initial database consists of wild-caught samples from 4 geographic locations as well as laboratory colony samples that originated from 7 additional locations using a genotyping by sequencing (GBS) approach. Geographic population structuring was identified for twelve populations that clustered according to geographic location. The Florida outbreak samples appeared similar to samples from the outer Caribbean cluster which included samples from Dominican Republic and Trinidad and Tobago, however, these results will be further clarified with the replacement of laboratory colony samples with future wild-caught samples.

The microbiome of wild and mass-reared new world screwworm, Cochliomyia hominivorax.

Insect population control through continual releases of large numbers of sterile insects, called sterile insect technique (SIT), is only possible if one can mass-rear large quantities of healthy insects. Adaptation of insect stocks to rearing conditions and artificial feeding systems can have a multitude of negative effects such as inbreeding depression, reduced compatibility with wild strains, unintentional selection for traits that lower fitness after release, and an altered microbiome. Changes to insect microbiomes can have many effects on insects ranging from a reduction in sex pheromones or reduced fitness. Thus understanding these systems is important for mass rearing and the performance of the sterile insect control programs. In this study we explored the microbiome of the New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) an economically important parasite of warm-blooded animals. Samples from myiases in cows and wild adults were compared to and mass-reared flies used by the SIT program. Significant differences were observed between these treatments, with wild captured flies having a significantly more diverse microbial composition. Bacteria known to stimulate oviposition were found in both wild and mass-reared flies. Two bacteria of veterinary importance were abundant in wild flies, suggesting screwworm is a potential vector of these diseases. Overall, this study provides the screwworm eradication program a platform to continue exploring the effects associated bacteria have on screwworm fitness.

Disruption of the odorant coreceptor Orco impairs foraging and host finding behaviors in the New World screwworm fly.

The evolution of obligate ectoparasitism in blowflies (Diptera: Calliphoridae) has intrigued scientists for over a century, and surprisingly, the genetics underlying this lifestyle remain largely unknown. Blowflies use odors to locate food and oviposition sites; therefore, olfaction might have played a central role in niche specialization within the group. In insects, the coreceptor Orco is a required partner for all odorant receptors (ORs), a major gene family involved in olfactory-evoked behaviors. Hence, we characterized the Orco gene in the New World screwworm, Cochliomyia hominivorax, a blowfly that is an obligate ectoparasite of warm-blooded animals. In contrast, most of the closely related blowflies are scavengers that lay their eggs on dead animals. We show that the screwworm Orco orthologue (ChomOrco) is highly conserved within Diptera, showing signals of strong purifying selection. Expression of ChomOrco is broadly detectable in chemosensory appendages, and is related to morphological, developmental, and behavioral aspects of the screwworm biology. We used CRISPR/Cas9 to disrupt ChomOrco and evaluate the consequences of losing the OR function on screwworm behavior. In two-choice assays, Orco mutants displayed an impaired response to floral-like and animal host-associated odors, suggesting that OR-mediated olfaction is involved in foraging and host-seeking behaviors in C. hominivorax. These results broaden our understanding of the chemoreception basis of niche occupancy by blowflies.

An early female lethal system of the New World screwworm, Cochliomyia hominivorax, for biotechnology-enhanced SIT.

BACKGROUND: The New World Screwworm fly (NWS), Cochliomyia hominivorax, is an ectoparasite of warm-blooded animals and a major pest of livestock in parts of South America and the Caribbean where it remains endemic. In North and Central America it was eradicated using the Sterile Insect Technique (SIT). A control program is managed cooperatively between the governments of the United States and Panama to prevent the northward spread of NWS from infested countries in South America. This is accomplished by maintaining a permanent barrier through the release of millions of sterile male and female flies in the border between Panama and Colombia. Our research team demonstrated the utility of biotechnology-enhanced approaches for SIT by developing a male-only strain of the NWS. The strain carried a single component tetracycline repressible female lethal system where females died at late larval/pupal stages. The control program can be further improved by removing females during embryonic development as larval diet costs are significant. RESULTS: The strains developed carry a two-component system consisting of the Lucilia sericata bottleneck gene promoter driving expression of the tTA gene and a tTA-regulated Lshid proapoptotic effector gene. Insertion of the sex-specifically spliced intron from the C. hominivorax transformer gene within the Lshid gene ensures that only females die when insects are reared in the absence of tetracycline. In several double homozygous two-component strains and in one "All-in-one" strain that had both components in a single construct, female lethality occurred at the embryonic and/or first instar larval stages when raised on diet without tetracycline. Laboratory evaluation for phenotypes that are relevant for mass rearing in a production facility revealed that most strains had fitness characteristics similar to the wild type J06 strain that is currently reared for release in the permanent barrier. Testing of an "All in one" strain under mass rearing conditions showed that the strain maintained the fitness characteristics observed in small-scale rearing. CONCLUSIONS: The early female lethal strains described here could be selected by the NWS Control Program for testing at large scale in the production facility to enhance the efficiency of the NWS eradication program.

Physiological and molecular correlates of the screwworm fly attraction to wound and animal odors.

The screwworm fly, Cochliomyia hominivorax (Coquerel), was successfully eradicated from the United States by the sterile insect technique (SIT). However, recent detection of these flies in the Florida Keys, and increased risk of introductions to the other areas warrant novel tools for management of the flies. Surveillance, a key component of screwworm control programs, utilizes traps baited with rotting liver or a blend of synthetic chemicals such as swormlure-4. In this work, we evaluated the olfactory physiology of the screwworm fly and compared it with the non-obligate ectoparasitic secondary screwworm flies, C. macellaria, that invade necrotic wound and feed on dead tissue. These two species occur in geographically overlapping regions. C. macellaria, along with other blowflies such as the exotic C. megacephala, greatly outnumber C. hominivorax in the existing monitoring traps. Olfactory responses to swormlure-4 constituents between sex and mating status (mated vs unmated) in both species were recorded and compared. Overall, responses measured by the antennograms offered insights into the comparative olfactory physiology of the two fly species. We also present detailed analyses of the antennal transcriptome by RNA-Sequencing that reveal significant differences between male and female screwworm flies. The differential expression patterns were confirmed by quantitative PCR. Taken together, this integrated study provides insights into the physiological and molecular correlates of the screwworm's attraction to wounds, and identifies molecular targets that will aid in the development of odorant-based fly management strategies.

Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs.

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.

Specific Gene Disruption in the Major Livestock Pests Cochliomyia hominivorax and Lucilia cuprina Using CRISPR/Cas9.

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 Chominivorax and Lcuprina 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 Chominivorax 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.

Semiochemicals released from five bacteria identified from animal wounds infested by primary screwworms and their effects on fly behavioral activity.

BACKGROUND: The Primary screwworm, Cochliomyia hominivorax (Coquerel), is a serious pest feeding on living flesh of any warm-blooded animal, including humans. It was eradicated from the United States in the early 1980s using the sterile male technique. However, it was recently detected in populations of wild deer and pets in the Florida Keys of the US. For monitoring purposes, screwworm flies are normally trapped using attractant bait with liver. However, there has been little effort to develop an efficient monitoring system for detection of screwworm flies using a specific synthetic attractant blend. Several studies have shown that odors from animal wound fluids attract screwworm adults, particularly gravid females. Bacteria associated with animal wounds have been identified that act as a major source for this attraction. To understand what volatiles attract screwworms we inoculated bovine blood with previously identified bacteria. We identified volatile chemicals released from the inoculated blood and other selected media over time and assessed the effect of those chemicals on behavioral activity of adult screwworm flies. METHODOLOGY/PRINCIPAL FINDINGS: A total of 7 volatile compounds were collected from bacteria incubated in either broth or blood using solid-phase microextraction, and their chemical structures were identified by their characteristic mass spectrum fragments and confirmed by retention times in comparison to those of synthetic standards via gas chromatograph combined mass spectrometry analyses. Five major volatiles including dimethyl disulfide, dimethyl trisulfide, phenol, p-cresol and indole were detected from a mixture of 5 bacteria incubated in blood. The ratios of volatiles released differed among different incubation media, time and individual bacteria. A synthetic mixture containing the five compounds was demonstrated to be attractive to adult screwworm flies both in laboratory assays and field trapping trials. CONCLUSIONS/SIGNIFICANCE: The results obtained from this study may assist in developing an efficient trapping system using the identified attractant blend to detect the infestation of primary screwworms. This is also the first study to explore the complex systems in volatile release profiles from 5 bacteria isolated from screwworm-infested animal wounds that are incubated with different media and incubation time, as well as individual and multi-species bacterial communities.

Distribution and Persistence of Sterile Screwworms (Diptera: Calliphoridae) Released at the Panama-Colombia Border.

The sterile insect technique is used by the Comisión Panamá - Estados Unidos para la Erradicación y Prevención del Gusano Barrenador del Ganado (COPEG) to maintain a barrier at the border of Panama and Colombia to prevent screwworms, Cochliomyia hominivorax (Coquerel), from South America reinfesting North America. Before studying the distribution and persistence of sterilized, mass-produced screwworms released in the barrier zone, the utility of applying fluorescent dust (∼1.0 mg/fly) to pupae and to newly emerged adults was evaluated to determine the potential effect on fly survival. The flight ability of flies collected from two adult emergence/collection systems (enclosed towers and open chambers) and treated with low (∼0.20 mg/fly) or high (∼1.0 mg/fly) amounts of fluorescent powder was compared. The distribution and persistence of sterile screwworms marked with fluorescent powder (∼0.20 mg/fly), after collection from the same two adult emergence/collection systems, was compared after their release in the barrier zone. The results demonstrated that: 1) fluorescent dust did not negatively affect sterile screwworm longevity or flight ability; 2) no differences were detected between sterile flies collected from the two emergence systems; and 3) sterile screwworms distributed evenly in the barrier zone and persisted for > 6 d. This information was useful in implementing the use of a new sterile fly emergence/collection system and deploying a new strain by COPEG for the barrier zone maintenance program; it will be valuable for evaluating alternative release strategies of sterile screwworms by the eradication and barrier maintenance program.

A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique.

BACKGROUND: The 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. RESULTS: Here, 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. CONCLUSIONS: We 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.

Managing Ammonia Emissions From Screwworm Larval Rearing Media.

Mass production, sterilization, and release of screwworms (Cochliomyia hominivorax (Coquerel)) that were competitive in the field significantly contributed to the successful application of the sterile insect technique for eradication of screwworms from continental North America. Metabolic byproducts resulting from protein-rich diets required for larval screwworms lead to ammonia liberation, sometimes at high levels, within the mass rearing facility. Until recently a sodium polyacrylate gel bulking agent was used for the larval media and adsorbed much of the ammonia. A need to replace the gel with an environmentally "friendly" bulking agent, while not increasing ammonia levels in the rearing facility, led to a series of experiments with the objective of developing procedures to reduce ammonia emissions from the larval media bulked with cellulose fiber. Additives of ammonia-converting bacteria, potassium permanganate, and Yucca schidigera Roezl ex Otrgies powder extract, previously reported to reduce ammonia levels in organic environments, were evaluated. Ammonia-converting bacteria did not have a positive effect. Addition of Y. schidigera powder extract (∼1% of total volume), potassium permanganate (∼250 ppm), and a combination of these two additives (at these same concentrations) kept ammonia at equivalent levels as when larval media was bulked with gel. Potassium permanganate also had sufficient antimicrobial properties that the use of formaldehyde in the diet was not necessary. Further testing is needed, at a mass rearing level, before full implementation into the screwworm eradication program.

Laboratory environment effects on the reproduction and mortality of adult screwworm (Diptera: Calliphoridae)

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 34.5°C), two diets (2 percent spray-dried blood plus 0.05 percent vitamins and corn syrup carrageenan) and three population densities (300, 400, and 500 flies/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 significantly greater when fed the protein diet and reared at 24.5°C and 34.5°C. Egg hatch was significantly less when the flies were reared at 34.5°C. When exposed to high temperatures (37°C and 40°C) 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.

A mosca-da-bicheira, Cochliomyia hominivorax Coquerel, é criada em escala massal com 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°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 significativamente maior quando alimentados com a dieta protéica e criados a 24,5°C e 34,5°C. A eclosão de larvas foi significativamente menor quando as moscas foram criadas a 34,5°C. Nas temperaturas mais elevadas (37°C e 40°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).

Laboratory environment effects on the reproduction and mortality of adult screwworm (Diptera: Calliphoridae).

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 34.5 degrees C), two diets (2% spray-dried blood plus 0.05% vitamins and corn syrup carrageenan) and three population densities (300, 400, and 500 flies/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 significantly greater when fed the protein diet and reared at 24.5 degrees C and 34.5 degrees C. Egg hatch was significantly less when the flies were reared at 34.5 degrees C. When exposed to high temperatures (37 degrees C and 40 degrees C) 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.