Ophthalmomyiasis Case Caused by Two Blow Fly (Diptera: Calliphoridae) Species in North America.

Ophthalmomyiasis is the result of fly larvae feeding on the tissues of the eye. Commonly associated with poor hygiene and open wounds, this condition is rare and often stigmatized. Treatment can be straightforward, and full recovery is common. Identifying the species responsible for ophthalmomyiasis is important for the medical, forensic, and entomological communities. Here, we present a case of ophthalmomyiasis where 30-40 blow fly (Diptera: Calliphoridae) larvae were removed from the eye of a human male. A representative subsample of five larvae was used for taxonomic identification via two approaches (a) DNA analysis, via sequencing of the complete mitochondrial genome (mtGenome) and comparison of the mtGenome and mitochondrial COI barcode region to GenBank, and (b) morphology, examination of the posterior spiracles using microscopy, and comparison to published larval descriptions of blow flies. Two species of blow flies were identified from the DNA analysis: Lucilia coeruleiviridis and Phormia regina. Morphological examination could only confirm L. coeruleiviridis as being present. To our knowledge, finding two blow fly species causing ophthalmomyiasis in a single individual has not been previously reported in the scientific literature. Neither P. regina nor L. coeruleiviridis prefers living tissue for larva development, but since they fill similar ecological niches, perhaps this was a show of competition rather than a normal feeding habit. Knowing these blow fly species can resort to this behavior, and that it can affect human populations, is valuable to the education of patients and providers.

DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples.

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies' DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples > alcohol-preserved old samples > untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.

Complete Mitochondrial Genome for Lucilia cuprina dorsalis (Diptera: Calliphoridae) from the Northern Territory, Australia.

The Australian sheep blowfly, Lucilia cuprina dorsalis, is a major sheep ectoparasite causing subcutaneous myiasis (flystrike), which can lead to reduced livestock productivity and, in severe instances, death of the affected animals. It is also a primary colonizer of carrion, an efficient pollinator, and used in maggot debridement therapy and forensic investigations. In this study, we report the complete mitochondrial (mt) genome of L. c. dorsalis from the Northern Territory (NT), Australia, where sheep are prohibited animals, unlike the rest of Australia. The mt genome is 15,943 bp in length, comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. The gene order of the current mt genome is consistent with the previously published L. cuprina mt genomes. Nucleotide composition revealed an AT bias, accounting for 77.5% of total mt genome nucleotides. Phylogenetic analyses of 56 species/taxa of dipterans indicated that L. c. dorsalis and L. sericata are the closest among all sibling species of the genus Lucilia, which helps to explain species evolution within the family Luciliinae. This study provides the first complete mt genome sequence for L. c. dorsalis derived from the NT, Australia to facilitate species identification and the examination of the evolutionary history of these blowflies.

Establishment of a multiplex PCR-CE assay for the simultaneous and rapid analysis of age markers for Calliphora vicina pupae.

Insects, especially blow flies, are forensically relevant to determine the minimal postmortem interval (PMImin), based on the fact that they are usually the first colonisers of a body. By estimating the age of immature blow flies, interferences can be made about the time since death. Whilst morphological parameters are valuable for age estimation of blow fly larvae, gene expression profiling is more applicable for blow fly pupae. Here, the age-dependent changes in the gene expression levels during the development are analysed. 28 temperature-independent markers have already been described for the age estimation of pupae of the forensically important blow fly Calliphora vicina and are analysed by RT-qPCR. To allow simultaneous analysis of these age markers, a multiplex assay was developed in the present study. After reverse transcription, the markers are analysed simultaneously in an endpoint PCR and subsequently separated by capillary electrophoresis (CE). This method is highly attractive due to its quick and easy procedure and interpretation. The present age prediction tool was adapted and validated. The multiplex PCR assay reproduced the same expression profiles as the RT-qPCR assay based on the same markers. The statistical evaluation shows that the new assay has a lower precision but a better trueness for age determination compared to the RT-qPCR assay. Since the new assay is also qualified to estimate the age of C. vicina pupae and is practical, cost-effective and, even more importantly, time-saving, it is attractive for use in forensic casework.

Identification and functional analysis of Cochliomyia hominivorax U6 gene promoters.

The New World screwworm, Cochliomyia hominivorax, is an obligate parasite, which is a major pest of livestock. While the sterile insect technique was used very successfully to eradicate C. hominivorax from North and Central America, more cost-effective genetic methods will likely be needed in South America. The recent development of CRISPR/Cas9-based genetic approaches, such as homing gene drive, could provide a very efficient means for the suppression of C. hominivorax populations. One component of a drive system is the guide RNA(s) driven by a U6 gene promoter. Here, we have developed an in vivo assay to evaluate the activity of the promoters from seven C. hominivorax U6 genes. Embryos from the related blowfly Lucilia cuprina were injected with plasmid DNA containing a U6-promoter-guide RNA construct and a source of Cas9, either protein or plasmid DNA. Activity was assessed by the number of site-specific mutations in the targeted gene in hatched larvae. One promoter, Chom U6_b, showed the highest activity. These U6 gene promoters could be used to build CRISPR/Cas9-based genetic systems for the control of C. hominivorax.

A short fragment of mitochondrial DNA for the taxonomic identification of blow flies (Diptera: Calliphoridae) in northwestern South America.

Blow flies are of medical, sanitary, veterinary, and forensic importance. Their accurate taxonomic identification is essential for their use in applied research. However, neotropical fauna has not been completely studied or described, and taxa identification without the required training is a difficult task. Additionally, the current morphological keys are not fitting to all extant taxa. Molecular-based approaches are widely used to overcome these issues, including the standard 5' COI barcode fragment (~650 base pairs [bp]) for identification at the species level. Here, a shorter sequence of 5' COI fragment (~342 bp) was assessed for the identification of 28 blow fly species inhabiting the northwest of South America. One tree-based (the generalized mixed Yule-coalescent-GMYC) and 3 distance-based approaches (automatic barcode gap discover - ABGD, the best close match - BCM, and the nearest neighbor - NN) analyses were performed. Noticeably, the amplification and sequencing of samples that had been preserved for up to 57 years were successful. The tree topology assigned 113 sequences to a specific taxon (70% effectiveness), while the distance approach assigned to 95 (59% effectiveness). The short fragment allowed the molecular identification of 19 species (60% of neotropical species except for the Lucilia species and Hemilucilia semidiaphana). According to these findings, the taxonomic and faunistic considerations of the blow fly fauna were provided. Overall, the short fragment approach constitutes an optimal species confirmation tool for the most common blow flies in northwestern South America.

Evidence of two mitochondrial lineages and genetic variability in forensically important Lucilia eximia (Diptera: Calliphoridae) in Colombia.

Lucilia eximia (Wiedemann, 1819) (Diptera: Calliphoridae) is a blowfly with medical and forensic importance that shows genetic and color variation, however, these variations have not justified the description of new species. But in forensic entomology an accurate identification of species and subpopulations is crucial. We explored the genetic variation of L. eximia from eight localities, in five natural regions in Colombia using two mitochondrial fragments, including the standard locus for insect identification COI and the Cytb-tRNA-Ser-ND1 region. We found significant differentiation at COI and Cytb-tRNA-Ser-ND1 level, characterizing two lineages and revealing a deep and significant genetic split. High values of FST and genetic distances supported the two lineages. The origin of the divergence of L. eximia remains to discover. Examining whether the lineages have diverse ecological and biological behaviors could be a significant impact on the use of L. eximia in forensic and medical science. Our results could have relevant implications for the use of post-mortem interval estimation based on insect evidence, as well as our sequences improve the database used in DNA-based methods for identifying forensically important flies.

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.

Development of the new microsatellite markers of Lucilia sericata (Diptera: Calliphoridae) from Korea.

BACKGROUND: Lucilia sericata is a medical and veterinary important insect species because its larvae feed on tissues of vertebrates including humans. Very few microsatellite makers have been reported from the species to illuminate its genetic variability and population genetic structure. METHODS AND RESULTS: In this study, L. sericata samples were collected from four different localities in Korea to develop the microsatellite markers to provide basic information on the genetic variability and population genetic structure in Korea of this species. In total, ten new microsatellite markers were sequenced and analyzed. Genetic diversity was performed using these microsatellite markers. The observed heterozygosity varied from 0.205 to 0.824, with an average of 0.546. The expected heterozygosity ranged from 0.579 to 0.886, with an average of 0.804. PIC value varied from 0.553 to 0.876. CONCLUSIONS: The markers developed in the present study are expected as informative for estimating genetic diversity of L. sericata.

Conditional knockdown of transformer in sheep blow fly suggests a role in repression of dosage compensation and potential for population suppression.

The transformer (tra) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina. Sex-specific tra RNA splicing is controlled by Sex lethal (Sxl) in Drosophila melanogaster but is auto-regulated in L. cuprina. Sxl also represses X chromosome dosage compensation in female D. melanogaster. We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L. cuprina. In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L. cuprina. However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos.

Age-dependent gene expression of Calliphora vicina pupae (Diptera: Calliphoridae) at constant and fluctuating temperatures.

Estimating the age of the developmental stages of the blow fly Calliphora vicina (Diptera: Calliphoridae) is of forensic relevance for the determination of the minimum post-mortem interval (PMImin). Fly eggs and larvae can be aged using anatomical and morphological characters and their modification during development. However, such methods can only hardly be applied for aging fly pupae. Previous study described age estimation of C. vicina pupae using gene expression, but just when reared at constant temperatures, but fluctuating temperatures represent a more realistic scenario at a crime scene. Therefore, age-dependent gene expression of C. vicina pupae were compared at 3 fluctuating and 3 constant temperatures, the latter representing the mean values of the fluctuating profiles. The chosen marker genes showed uniform expression patterns during metamorphosis of C. vicina pupae bred at different temperature conditions (constant or fluctuating) but the same mean temperature (e.g. constant 10 °C vs. fluctuating 5-15 °C). We present an R-based statistical tool, which enables estimation of the age of the examined pupa based on the analysed gene expression data.

Evaluation of Reference Genes and Age Estimation of Forensically Useful Aldrichina grahami (Diptera: Calliphoridae) During Intrapuparial Period.

The minimum postmortem interval (PMImin) could be evaluated from the developmental stage of forensically important insects colonize a corpse, such as blow flies (Diptera: Calliphoridae). Unlike larvae, the developmental stage of which is well established according to their morphology, estimating the age of pupae is proven to be challenging. Recently, several studies reported the regulation of special genes during the development of blow fly pupae. However, gene regulation in Aldrichina grahami during the intrapuparial period remains to be studied. Therefore, we set out to investigate the mRNA levels of heat shock protein 23 (Hsp23), heat shock protein 24 (Hsp24), and 1_16 during the metamorphosis of A. grahami pupae. First, we examined seven candidate reference genes (ribosomal protein 49 (RP49), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), beta-tubulin at 56D (ß-tubulin), Ribosomal protein L23 (RPL23), glutathione S-transferase (GST1), and Actin. Three widely used algorithms (NormFinder, BestKeeper, and geNorm) were applied to evaluate the mRNA levels of reference gene candidates in puparium at three stable temperatures (15, 22, and 27°C). Next, mRNA expression of Hsp23, Hsp24, and 1_16 during A. grahami metamorphosis was examined. We demonstrated that mRNA expression levels of Hsp23, Hsp24, and 1_16 showed time-specific regulation. In summary, our study identified three gene markers for the intrapuparial period of A. grahami and might provide a potential application in PMImin estimation.

Evolution of coding sequence and gene expression of blowflies and botflies with contrasting feeding habits.

The Oestroidea superfamily is characterized by the diversity of feeding preferences among closely-related species; these flies are saprophagous, obligate parasites, or facultative parasites. We used gene expression and coding sequence data from five species (Cochliomyia hominivorax, Chrysomya megacephala, Lucilia cuprina, Dermatobia hominis, and Oestrus ovis) to identify underlying genetic differences involved in the diverse lifestyles. We tested whether 1287 orthologs have different expression and evolutionary constraints under different scenarios. We found two up-regulated genes; one in species causing cutaneous myiasis that is involved in iron transportation/metabolization (ferritin), and another in species causing traumatic myiasis that responds to reduced oxygen levels (anoxia up-regulated-like). Our evolutionary analysis showed a similar result. In the Co. hominivorax branch, we found one gene with the same function as ferritin that may be evolving under positive selection, spook. This is the first step towards understanding origins and evolution of parasitic strategy diversity in Oestroidea.

Building a transgenic sexing strain for genetic control of the Australian sheep blow fly Lucilia cuprina using two lethal effectors.

BACKGROUND: The sterile insect technique (SIT) has been successfully used in many pest management programs worldwide. Some SIT programs release both sexes due to the lack of genetic sexing strains or efficient sex separation methods but sterile females are ineffective control agents. Transgenic sexing strains (TSS) using the tetracycline-off control system have been developed in a variety of insect pests, from which females die by either of two commonly used lethal effectors: overexpression of the transcription factor tetracycline transactivator (tTA) or ectopic expression of a proapoptotic gene, such as head involution defective (hid). The lethality from tTA overexpression is thought to be due to "transcriptional squelching", while hid causes lethality by induction of apoptosis. This study aims to create and characterize a TSS of Lucilia cuprina, which is a major pest of sheep, by combining both lethal effectors in a single transgenic strain. RESULTS: Here a stable TSS of L. cuprina (DH6) that carries two lethal effectors was successfully generated, by crossing FL3#2 which carries a female-specific tTA overexpression cassette, with EF1#12 which carries a tTA-regulated LshidAla2 cassette. Females with one copy of the FL3#2 transgene are viable but up to 99.8% of homozygous females die at the pupal stage when raised on diet that lacks tetracycline. Additionally, the female lethality of FL3#2 was partially repressed by supplying tetracycline to the parental generation. With an additional LshidAla2 effector, the female lethality of DH6 is 100% dominant and cannot be repressed by maternal tetracycline. DH6 females die at the late-larval stage. Several fitness parameters important for mass rearing such as hatching rate, adult emergence and sex ratio were comparable to those of the wild type strain. CONCLUSIONS: Compared to the parental FL3#2 strain, the DH6 strain shows stronger female lethality and lethality occurs at an earlier stage of development. The combination of two tTA-dependent lethal effectors could improve strain stability under mass rearing and could reduce the risk of resistance in the field if fertile males are released. Our approach could be easily adapted for other pest species for an efficient, safe and sustainable genetic control program.

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.

The evolution of sexually dimorphic cuticular hydrocarbons in blowflies (Diptera: Calliphoridae).

Cuticular hydrocarbons (CHCs) are organic compounds found on the cuticles of all insects which can act as close-contact pheromones, while also providing a hydrophobic barrier to water loss. Given their widespread importance in sexual behaviour and survival, CHCs have likely contributed heavily to the adaptation and speciation of insects. Despite this, the patterns and mechanisms of their diversification have been studied in very few taxa. Here, we perform the first study of CHC diversification in blowflies, focussing on wild populations of the ecologically diverse genus Chrysomya. We convert CHC profiles into qualitative and quantitative traits and assess their inter- and intra-specific variation across 10 species. We also construct a global phylogeny of Chrysomya, onto which CHCs were mapped to explore the patterns of their diversification. For the first time, we demonstrate that blowflies express an exceptional diversity of CHCs, which have diversified in a nonphylogenetic and punctuated manner, are species-specific and sexually dimorphic. It is likely that both ecological and sexual selection have shaped these patterns of CHC diversification, and our study now provides a comprehensive framework for testing such hypotheses.

Wound healing potential: evaluation of molecular profiling and amplification of Lucilia sericata angiopoietin-1 mRNA mid-part.

OBJECTIVE: High prevalence of chronic ulcers and the burden of disease necessitate the increasingly significant production of new recombinant proteins in the world. The angiopoietin-1 enzyme is a part of the growth factors group which is secreted by Lucilia sericata (Diptera: Calliphoridae) larvae when they meet lesions to ensure maggot therapy. It is one of the most potent proteins in wound healing. Given its essential role, the angiopoietin-1 gene of L. sericata was characterized, which provided some necessary information on its identity. RESULTS: The mid-part of the angiopoietin-1 mRNA sequence was thus characterized based on the design of different primers such as exon-exon junction, conserved regions, and specific region primers via conventional polymerase chain reaction (PCR). Its structural features were configured by in silico method. The sequence of mid-part (390 bp) of angiopoietin-1 was determined empirically, and BLAST analysis unraveled its high identity (85%) with the sequence of angiopoietin-1 mRNA of the larval housefly, Musca domestica. The homology of this enzyme also exhibited that its nucleic acid sequence was very similar to the domains of angiopoietin-1 in Lucilia cuprina. The current data are instructive and critical to evaluate the action of this enzyme in recombinant protein production in future molecular studies on wound healing.

An Evaluation of Differentially Spliced Genes as Markers of Sex for Forensic Entomology,.

Blow flies (Calliphoridae) are important medically and economically and are commonly used in forensics as temporal markers in death investigations. While phenotypic traits in adult flies can be sexually dimorphic, sex identification in immatures is difficult. Consequently, little is known about how sex may result in developmental disparities among sexes even though there are indications that they may be important in some instances. Since genetic mechanisms for sex are well studied in model flies and species of agricultural and medical importance, we exploit the sex-specifically spliced genes transformer (tra) and doublesex (dsx) in the sex determination pathway to optimize a sex identification assay for immatures. Using known primer sets for tra and with a novel one for dsx, we develop PCR assays for identifying sex in four forensically relevant Calliphoridae species: Lucilia sericata (Meigen), Lucilia cuprina (Wiedemann), Cochliomyia macellaria (Fabricius), and Chrysomya rufifacies (Macquart) and evaluated their performance. Band detection rates were found to range from 71 to 100%, call rates ranged from 90 to 100%, and no error was found when bands could be called. Such information is informative for purposes of testimony and in preparation for development studies. The developed assays will assist in further differentiating sexually dimorphic differences in development of the Calliphoridae and aid in more accurately estimating insect age when age predictive markers (size, development time, molecular expression) are sexually dimorphic.

Chromosome-level genome assembly of Aldrichina grahami, a forensically important blowfly.

BACKGROUND: Blowflies (Diptera: Calliphoridae) are the most commonly found entomological evidence in forensic investigations. Distinguished from other blowflies, Aldrichina grahami has some unique biological characteristics and is a species of forensic importance. Its development rate, pattern, and life cycle can provide valuable information for the estimation of the minimum postmortem interval. FINDINGS: Herein we provide a chromosome-level genome assembly of A. grahami that was generated by Pacific BioSciences sequencing platform and chromosome conformation capture (Hi-C) technology. A total of 50.15 Gb clean reads of the A. grahami genome were generated. FALCON and Wtdbg were used to construct the genome of A. grahami, resulting in an assembly of 600 Mb and 1,604 contigs with an N50 size of 1.93 Mb. We predicted 12,823 protein-coding genes, 99.8% of which was functionally annotated on the basis of the de novo genome (SRA: PRJNA513084) and transcriptome (SRA: SRX5207346) of A. grahami. According to the co-analysis with 11 other insect species, clustering and phylogenetic reconstruction of gene families were performed. Using Hi-C sequencing, a chromosome-level assembly of 6 chromosomes was generated with scaffold N50 of 104.7 Mb. Of these scaffolds, 96.4% were anchored to the total A. grahami genome contig bases. CONCLUSIONS: The present study provides a robust genome reference for A. grahami that supplements vital genetic information for nonhuman forensic genomics and facilitates the future research of A. grahami and other necrophagous blowfly species used in forensic medicine.