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.

A new formulation of screwworm fly attractant with reduced hazardous chemicals and transport restrictions.

New World screwworm flies, Cochliomyia hominivorax (Coquerel), are obligate parasites of warm-blooded animals. They were eradicated from North and Central America during the mid-20th to early-21st centuries using the sterile insect technique (SIT), a method presently employed to maintain a permanent barrier between Central and South America. Lures are an important component of the screwworm eradication program, where they are used for surveillance, sample collection, and strain evaluation in the field. The first chemical lure, later named swormlure, was developed based on the attractiveness of C. hominivorax to volatile organic compounds (VOCs) produced from decomposing animal tissues. The formulation has changed little over the years and presently contains 10 chemicals, one of which is dimethyl disulfide (DMDS). Restrictions on the transport of DMDS have recently impeded its use in swormlure-4 (SL-4). However, dimethyl trisulfide (DMTS) is not as highly restricted and can be shipped via air transportation. Both chemicals are produced by microbial decomposition of animal tissues. Here, we conducted field trials using three releases of sterile C. hominivorax, each comprising approximately 93,000 flies, to test the efficacy of SL-4, containing DMDS, to swormlure-5 (SL-5) containing DMTS. Traps baited with SL-4 and SL-5 captured 575 (mean = 191.7, SD 17.9) and 665 (mean = 221.7, SD 33.2) C. hominivorax, respectively (df = 19, F = 1.294, P = 0.269). However, traps baited with SL-5 captured considerably more Cochliomyia macellaria (Fabricius), a closely related but nontarget fly.

Development of a diagnostic single nucleotide polymorphism (SNP) panel for identifying geographic origins of Cochliomyia hominivorax, the New World screwworm.

The New World screwworm, Cochliomyia hominivorax, causes myiasis in livestock, humans, and other warm-blooded animals in much of South America and the Caribbean. It has been eradicated from North and Central America using the sterile insect technique and a biological barrier is currently maintained at the Panama - Colombian border. However, C. hominivorax is still a threat to eradicated areas as outbreaks can and do occur. In order to identify the origin of a fly involved in an outbreak scenario, diagnostic tools would be beneficial. Recently, the geographic population structure of this species was identified using single nucleotide polymorphisms (SNPs). Here we characterize the three major regional clusters: South America, the Inner Caribbean, and the Outer Caribbean. The objective of this study was to develop a SNP (single nucleotide polymorphism) panel to distinguish between these three clusters. A panel was developed using two unique SNPs per region for a total of six SNPs. This diagnostic SNP assay will allow for rapid source determination of flies from future incursions in order to intercept introductory pathways and aid in the control of New World screwworm.

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.

New World screwworm (Cochliomyia hominivorax) myiasis in feral swine of Uruguay: One Health and transboundary disease implications.

BACKGROUND: Feral swine (Sus scrofa) are highly invasive and threaten animal and human health in the Americas. The screwworm (Cochliomyia hominivorax) is listed by the World Organization for Animal Health as a notifiable infestation because myiasis cases affect livestock, wildlife, and humans in endemic areas, and outbreaks can have major socioeconomic consequences in regions where the screwworm has been eradicated. However, a knowledge gap exists on screwworm infestation of feral swine in South America, where the screwworm is endemic. Here, we report screwworm infestation of feral swine harvested in Artigas Department (Uruguay), where the Republic of Uruguay shares borders with Brazil and Argentina. METHODS: Myiasis caused by the larvae of screwworm were identified in feral swine with the support and collaboration of members of a local feral swine hunting club over a 3-year period in the Department of Artigas. Harvested feral swine were examined for the presence of lesions where maggots causing the myiasis could be sampled and processed for taxonomic identification. The sites of myiasis on the body of infested feral swine and geospatial data for each case were recorded. The sex and relative size of each feral swine were also recorded. Temperature and precipitation profiles for the region were obtained from public sources. RESULTS: Myiases caused by screwworms were recorded in 27 of 618 the feral swine harvested. Cases detected in males weighing > 40 kg were associated with wounds that, due to their location, were likely caused by aggressive dominance behavior between adult males. The overall prevalence of screwworm infestation in the harvested feral swine was associated with ambient temperature, but not precipitation. Case numbers peaked in the warmer spring and summer months. CONCLUSIONS: This is the first report on myiasis in feral swine caused by screwworm in South America. In contrast to myiasis in cattle, which can reach deep into host tissues, screwworms in feral swine tended to cause superficial infestation. The presence of feral swine in screwworm endemic areas represents a challenge to screwworm management in those areas. Screwworm populations maintained by feral swine may contribute to human cases in rural areas of Uruguay, which highlights the importance of the One Health approach to the study of this invasive host species-ectoparasite interaction.

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.

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.

Innate immune system capabilities of the Asian citrus psyllid, Diaphorina citri.

Citrus production worldwide is currently threatened by Huanglongbing, or citrus greening disease. The associated pathogen, Candidatus Liberibacter asiaticus (CLas), is transmitted by the Asian citrus psyllid, Diaphorina citri. Annotation of the D. citri genome revealed a reduced innate immune system lacking a number of antimicrobial peptides and the Imd pathway associated with defense against Gram-negative bacteria. We characterized this apparent immune reduction in survival assays in which D. citri were exposed to Gram-negative or Gram-positive bacteria. D. citri experienced significant mortality when exposed to Serratia marcescens (Gram-negative) through oral ingestion or by septic injury. Escherichia coli (Gram-negative) also caused significant D. citri mortality, but only when inoculated at high concentrations through oral ingestion or by septic injury. Neither Micrococcus luteus (Gram-positive) or Bacillus subtilis (Gram-positive) caused significant mortality as compared to controls in any experiment. E. coli titers increased rapidly following exposure, while M. luteus titer remained stable for 72 h. We demonstrate that D. citri is capable of defending against E. coli, a Gram-negative bacterium, despite lacking the Imd defense pathway. The tolerance of D. citri to M. luteus infection, yet inability to effectively clear infections, presents questions to efficacy of D. citri immune response to effectively clear Gram-positive infections.

Annotation of the Asian Citrus Psyllid Genome Reveals a Reduced Innate Immune System.

Citrus production worldwide is currently facing significant losses due to citrus greening disease, also known as Huanglongbing. The citrus greening bacteria, Candidatus Liberibacter asiaticus (CLas), is a persistent propagative pathogen transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). Hemipterans characterized to date lack a number of insect immune genes, including those associated with the Imd pathway targeting Gram-negative bacteria. The D. citri draft genome was used to characterize the immune defense genes present in D. citri. Predicted mRNAs identified by screening the published D. citri annotated draft genome were manually searched using a custom database of immune genes from previously annotated insect genomes. Toll and JAK/STAT pathways, general defense genes Dual oxidase, Nitric oxide synthase, prophenoloxidase, and cellular immune defense genes were present in D. citri. In contrast, D. citri lacked genes for the Imd pathway, most antimicrobial peptides, 1,3-ß-glucan recognition proteins (GNBPs), and complete peptidoglycan recognition proteins. These data suggest that D. citri has a reduced immune capability similar to that observed in A. pisum, P. humanus, and R. prolixus. The absence of immune system genes from the D. citri genome may facilitate CLas infections, and is possibly compensated for by their relationship with their microbial endosymbionts.

A global comparison of Bactericera cockerelli (Hemiptera: Triozidae) microbial communities.

The potato psyllid (Bactericera cockerelli Sulc) is an economically important insect pest of solanaceous crops such as potato, tomato, pepper, and tobacco. Historically, the potato psyllid's range included central United States, Mexico, and California; more recently, populations of this insect have been reported in Central America, the Pacific Northwest, and New Zealand. Like most phytophagous insects, potato psyllids require symbiotic bacteria to compensate for nutritional deficiencies in their diet. Potato psyllids harbor the primary symbiont, Candidatus Carsonella ruddii, and may also harbor many secondary symbionts such as Wolbachia sp., Sodalis sp., Pseudomonas sp., and others. These secondary symbionts can have an effect on reproduction, nutrition, immune response, and resistances to heat or pesticides. To identify regional differences in potato psyllid bacterial symbionts, 454 pyrosequencing was performed using generic 16S rRNA gene primers. Analysis was performed using the Qiime 1.6.0 software suite, ARB Silva, and R. Operational taxonomic units were then grouped at 97% identity. Representative sequences were classified to genus using the ARB SILVA database. Potato psyllids collected in California contained a less diverse microbial community than those collected in the central United States and Central America. The crop variety, collection year, and haplotype did not seem to affect the microbial community in potato psyllids. The primary difference between psyllids in different regions was the presence and overall bacterial community composition of Candidatus Carsonella ruddii and Wolbachia.

Low-level detection of candidatus Liberibacter solanacearum in Bactericera cockerelli (Hemiptera: Triozidae) by 16s rRNA Pyrosequencing.

Accurate detection and quantification of Candidatus Liberibacter solanacearum (CLs), the putative causal agent of zebra chip disease of potato (Solanum tuberosum L.), in the potato psyllid, Bactericera cockerelli (Sulc), has become necessary to better understand the biology of the disease cycle. Studies on the transmission efficiency of potato psyllids have shown inconsistencies with field surveys. There have also been reports of laboratory colonies inexplicably losing and regaining CLs infection as detected by polymerase chain reaction (PCR). Until now, DNA primers were used to detect CLs in potato psyllid tissue using conventional polymerase chain reaction (PCR) and gel electrophoresis or by real-time quantitative PCR. In this study, CLs was detected using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) at levels identifiable by PCR, and low levels, including samples with only one cell of CLs. Potato psyllids with <300 pyrosequencing reads did not show positive using conventional PCR. These results indicate that the currently accepted PCR diagnostic technique produces false negatives due to detection limits higher than what is generally present in field collected psyllids, and also provides an explanation as to why laboratory colonies seem to lose and regain CLs infection.