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.

Transcriptomic profiling of different developmental stages reveals parasitic strategies of Wohlfahrtia magnifica, a myiasis-causing flesh fly.

BACKGROUND: Wohlfahrtia magnifica is an obligatory parasite that causes myiasis in several warm-blooded vertebrates. Adult females deposit the first-stage larvae directly onto wounds or natural body orifices (e.g., genitalia) of the host, from where they quickly colonize the host tissue and feed on it for development. The infestation of W. magnifica can lead to health issues, welfare concerns, and substantial economic losses. To date, little is known about the molecular mechanisms of the W. magnifica-causing myiasis. RESULTS: In this study, we collected parasitic-stage larvae of W. magnifica from wounds of naturally infested Bactrian camels, as well as pupae and adult flies reared in vitro from the wound-collected larvae, for investigating the gene expression profiles of the different developmental stages of W. magnifica, with a particular focus on examining gene families closely related to the parasitism of the wound-collected larvae. As key proteins related to the parasite-host interaction, 2049 excretory/secretory (ES) proteins were identified in W. magnifica through the integration of multiple bioinformatics approaches. Functional analysis indicates that these ES proteins are primarily involved in cuticle development, peptidase activity, immune response, and metabolic processes. The global investigation of gene expression at different developmental stages using pairwise comparisons and weighted correlation network analysis (WGCNA) showed that the upregulated genes during second-stage larvae were related to cuticle development, peptidase activity, and RNA transcription and translation; during third-stage larvae to peptidase inhibitor activity and nutrient reservoir activity; during pupae to cell and tissue morphogenesis and cell and tissue development; and during adult flies to signal perception, many of them involved in light perception, and adult behavior, e.g., feeding, mating, and locomotion. Specifically, the expression level analysis of the likely parasitism-related genes in parasitic wound-collected larvae revealed a significant upregulation of 88 peptidase genes (including 47 serine peptidase genes), 110 cuticle protein genes, and 21 heat shock protein (hsp) genes. Interestingly, the expression of 2 antimicrobial peptide (AMP) genes, including 1 defensin and 1 diptericin, was also upregulated in the parasitic larvae. CONCLUSIONS: We identified ES proteins in W. magnifica and investigated their functional distribution. In addition, gene expression profiles at different developmental stages of W. magnifica were examined. Specifically, we focused on gene families closely related to parasitism of wound-collected larvae. These findings shed light on the molecular mechanisms underlying the life cycle of the myiasis-causing fly, especially during the parasitic larval stages, and provide guidance for the development of control measures against W. magnifica.

Genetic diversity of populations of Old World screwworm fly, Chrysomya bezziana, causing traumatic myiasis of livestock in the Gulf region and implications for control by sterile insect technique.

Fly larvae were collected from 181 cases of traumatic myiasis in livestock in 10 regions of four countries in the Middle East Gulf region: Iran, Iraq, Saudi Arabia and Oman. The predominant fly species responsible for cases was the Old World screwworm (OWS) fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae). In cases from Iran and Oman, which included non-OWS fly species, OWS fly was found solely responsible for 67.6% of cases and jointly with other fly species for a further 12.7% of cases. The major hosts were sheep and goats, together comprising 84.6% of the total, which reflects their predominance among the livestock of these Gulf countries. The major site of wounding on sheep and goats was the tail (40.3%), followed by female genitalia (14.0%). The 3' terminal 715 nucleotides of the mitochondrial cytochrome b gene were sequenced for 178 larvae of OWS. Five haplotypes were identified: three had been recorded previously in the region (two were common throughout and one was unique to Oman), and two were newly identified, one from southern Iraq and the other from Saudi Arabia, both in regions sampled for the first time. The haplotypes varied from one another only at one or two nucleotide sites, equivalent to an intraspecific difference of 0.14-0.28% across the entire 715-bp fragment. There was a single statistically significant association between host species and haplotype in Saudi Arabia, a first such record for OWS fly. The small degree of genetic diversity between geographical populations of OWS fly within the Gulf region suggests that a single Gulf colony could be used to implement the sterile insect technique within an integrated control programme.

Morphological and mitochondrial DNA characters for identification and phylogenetic analysis of the myiasis-causing flesh fly Wohlfahrtia magnifica and its relatives, with a description of Wohlfahrtia monegrosensis sp. n. Wyatt & Hall.

Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae) is a major cause of traumatic myiasis in livestock in Central and Eastern Europe and in countries bordering the Mediterranean. The present study explored the utility of external body characters, genitalia characters and mitochondrial DNA characters for identification of this and related species in the subfamily Paramacronychiinae. Sequence analyses of the 3' terminal 273 bp of the mitochondrial cytochrome b gene revealed two lineages of W. magnifica, one from Spain and France and the other from the rest of Eurasia, differing by only two base pairs. Phylogenetic analysis of cytochrome b showed that W. magnifica and Wohlfahrtia vigil Walker were sister species; this conclusion was not contradicted by a phylogenetic analysis of the morphological characters. Based on cytochrome b, the genetic distance between specimens of W. vigil from Europe and North America was sufficiently large to justify the recognition of more than one species. A new species, Wohlfahrtia monegrosensis, from northern Spain, was described, based on morphology and cytochrome b. A unique combination of external body characters of males or females were diagnostic for W. magnifica, the W. vigil group and Wohlfahrtia bella, but only the genitalia characters were diagnostic for all nine species studied.

Photographic map of the polytene chromosomes of Cochliomyia hominivorax.

Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. A polytene chromosome map is an invaluable tool for the genetic analysis and manipulation of any species because it allows the integration of physical and genetic maps. Cochliomyia hominivorax has a diploid number of 12 chromosomes (2n = 12): five pairs of autosomes and one pair of sex chromosomes (XX/XY), which do not polytenize. We created a new photomap of the polytene chromosomes of C. hominivorax describing its five autosomes (chromosomes 2-6). Pupal trichogen cells, which have chromosomes with a high degree of polytenization, were used to elaborate this map. The photomap was made by comparing 20 different nuclei and choosing, for each chromosome segment, the region with the highest resolution. Thus, we present a new photomap of the five autosomes of this species, with a total resolution of 1450 bands.

Parasite intensity and fur coloration in reindeer calves - contrasting artificial and natural selection.

1. In reindeer, the variation in fur coloration is higher in semi-domesticated than in wild populations. This difference might result from impacts of natural enemies acting on conspicuously coloured individuals with higher intensity in natural than semi-domesticated populations. 2. While predator pressure is rather similar for wild and semi-domesticated populations, semi-domesticated reindeer are less impacted by parasitism due to endectocidic treatment. In this study, we estimated the intensity of warble flies in different reindeer colour morphs in a large sample of untreated calves from multiple semi-domesticated herds in northern Norway. 3. We found that lighter coloured reindeer calves have higher intensities of warble fly larvae than darker ones. This is associated with a decrease in body mass, and may hence influence subsequent fitness of the animals. 4. The high intensity of parasites in white calves implies that they are either more exposed or less resistant to parasitic infections. In either case, parasitism by warble flies appears to be a proximate cause driving coloration of reindeer towards a reduced variability in natural populations.

Genetic approaches for studying myiasis-causing flies: molecular markers and mitochondrial genomics.

"Myiasis-causing flies" is a generic term that includes species from numerous dipteran families, mainly Calliphoridae and Oestridae, of which blowflies, screwworm flies and botflies are among the most important. This group of flies is characterized by the ability of their larvae to develop in animal flesh. When the host is a live vertebrate, such parasitism by dipterous larvae is known as primary myiasis. Myiasis-causing flies can be classified as saprophagous (free-living species), facultative or obligate parasites. Many of these flies are of great medical and veterinary importance in Brazil because of their role as key livestock insect-pests and vectors of pathogens, in addition to being considered important legal evidence in forensic entomology. The characterization of myiasis-causing flies using molecular markers to study mtDNA (by RFLP) and nuclear DNA (by RAPD and microsatellite) has been used to identify the evolutionary mechanisms responsible for specific patterns of genetic variability. These approaches have been successfully used to analyze the population structures of the New World screwworm fly Cochliomyia hominivorax and the botfly Dermatobia hominis. In this review, various aspects of the organization, evolution and potential applications of the mitochondrial genome of myiasis-causing flies in Brazil, and the analysis of nuclear markers in genetic studies of populations, are discussed.

[Forensic applications of the sequencing of mitochondrial DNA cytochrome oxidase subunit I and II gene for idenfication of Sarcosaphagous flies (diptera)].

OBJECTIVE: This study was based on a 278 and 635 bp region of the gene for cytochrome oxidase subunit I and I (CO I and CO II ) encoding region of mtDNA; the aim was to solve the problems in identifying Sarcosaphagous flies, particularly in the flies' larvae and eggs which could not be identified only by use of their morphologyical features. METHODS: Samples of sarcosaphagous flies and larvae were collected from those on the corps of rabbits on the grassland in the Huhhot district and of a pig on the sandy ground in the Dunhuang district. The mtDNA of flies and their larvae and eggs was extracted using the Chelex technique. Polymerase chain reactions were conducted on a Perkin-Elmer 9600 thermal cycler, followed by vertical non-denaturing polyacrylamide gelectrophoresis. PCR products were purified using the Nucleic Acid Purification Kit. Sequences of both strands were obtained by direct sequencing of the double-stranded PCR product using one of the PCR primers and the ABI PRISM Big Dye Terminator Cycle Sequencing Kit. Sequence reactions were electrophoresed on ABI Model 377 DNA Sequencers. A neighbour-joining tree using the Tamura and Nei model of nucleotide substitution was constructed using the MEGA2. 1 package. RESULTS: A 278 and 635 bp region of the gene for CO I and CO II encoding region of mtDNA of Sarcosaphagous flies and their larvae and eggs was noted to show the percentages for the sequence divergence within species (less than 1%) and the sequence divergence between species (above 3%). For species that diverged from all others by a relatively large percentage and had small within-species variation, the least percentages of sequence divergence were given which distinguished any individual within that species from any other species. CONCLUSION: A 278 and 635 bp region of the gene for CO I and CO II encoding region of mtDNA of Sarcosaphagous flies and their larvae and eggs has been effectively used for the molecular phylogeny and the identification of their species group. CO I and CO II, or CO II, is better than CO I.

Evolution and structural organisation of mitochondrial DNA control region of myiasis-causing flies.

This study reports the molecular characterization of the mtDNA control region (called the A+T-rich region in insects) of five dipteran species which cause myiasis: Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, Chrysomya megacephala Fabricius, Lucilia eximia Wiedemann (Diptera: Calliphoridae) and Dermatobia hominis Linnaeus Jr (Diptera: Oestridae). The control region in these species varies in length from 1000 to 1600 bp. Two structural domains with specific evolutionary patterns were identified. These were (1) conserved sequence blocks containing primary sequence motifs, including dinucleotide pyrimidine-purine series and long T-stretches, located at the 5' end adjacent to the tRNA(Ile) gene and (2) a hypervariable domain at the 3' end characterized by increased nucleotide divergence and size variation. A high frequency of A<-->T transversions at nucleotide substitution level indicated directional mutation pressure. The phylogenetic usefulness of the insect control region is discussed.

Breeding for disease resistance in Merino sheep in Australia.

Breeding for disease resistance in Merino sheep in Australia has attracted considerable research and development attention. Increased labour costs, the reduced efficacy of common anthelmintics and insecticides, consumer demand for products which are free of chemicals and the poor prognosis of alternative long-term control strategies are all forcing sheep breeders to contemplate the best animal health options available, including selective breeding for resistance. The three major diseases which affect sheep production include gastrointestinal nematode parasites, flystrike (cutaneous myiasis) and footrot. Genetic improvement has been shown to be feasible in all these diseases, possibly with little adverse effect on genetic progress in other production traits. Programmes for resistance to internal parasites to be included in practical breeding programmes are now in progress. This paper deals with the incentives for focusing on the three major diseases in Merino sheep, the potential sources of genetic variation, and the means to exploit these sources of variation. The authors also highlight gains and benefits achieved in experimental selection flocks, and the difficulties and options available for commercial breeders.

Cellular inflammatory responses in skin of sheep selected for resistance or susceptibility to fleece rot and fly strike.

Leucocyte populations were examined in normal and inflamed skin of sheep bred for resistance (R) or susceptibility (S) to bacterial fleece rot and the common sequela, body strike caused by the dipteran parasite Lucilia cuprina. No differences between R and S lines were found in numbers of neutrophils accumulating in acute inflammatory lesions induced by activated complement, leukotriene B4, interleukin (IL)-1 beta, tumour necrosis factor-alpha, IL-8 or endotoxin from Pseudomonas aeruginosa. T19+ (alpha gamma delta T cell subset) lymphocytes and eosinophils were more prevalent in skin of sheep from the S line whereas IgE+ cells were more prevalent in skin of sheep from the R line. In an unrelated population of sheep, animals with low fleece rot scores had more intense neutrophil migration into inflammatory lesions induced by all the mediators examined than did animals with high fleece rot scores. IgE+ cells were more prevalent in animals with low fleece rot scores, although in contrast to R and S lines, T19+ cells tended to be elevated in this group of animals. The results suggest that defence mechanisms associated with IgE+ cells in skin may play an important role in resistance to fleece rot and fly strike.

Plasma leakage in skin of sheep selected for resistance or susceptibility to fleece rot and fly strike.

The leakage of plasma into skin following injection of histamine, bradykinin, activated complement, platelet-activating factor and serotonin was measured in sheep bred for resistance or susceptibility to fleece rot and fly strike. Genetically susceptible sheep had significantly greater plasma leakage to activated complement than genetically resistant sheep, and for all mediators there was a trend for plasma leakage to be greater in susceptible sheep. Within each genotype there was also a tendency for plasma leakage to be positively correlated with fleece rot score. In a flock of sheep of different genetic background not selected for resistance or susceptibility to fleece rot and fly strike, positive phenotypic correlations were also noted between fleece rot and plasma leakage. Plasma leakage provides nutrition for the first instar larvae of Lucilia cuprina, the major cause of primary blowfly strike in Australia. Diminished leakage of plasma following release of endogenous permeability mediators may be one component of the mechanism that confers resistance in animals bred for resistance to fleece rot and fly strike.

Observations on fibre diameter variation of sheep in relation to fleece-rot and body strike susceptibility.

A comparative study of various fleece properties known to influence fleece-rot susceptibility was made in a merino flock consisting of sheep which were found to be either resistant or susceptible to fleece-rot and body strike following heavy rains. The fleece properties measured were fibre diameter, fibre diameter, fibre diameter variation, wax content, suint content, wax to suint ratio, suint pH, insoluble nitrogen content, wool colour and wettability. Fibre diameter variation, due mainly to the presence of coarse, secondary fibres in the staple, was the only fleece property which differed significantly (p less than 0.001) between resistant and susceptible animals. The coefficient of variation of fibre diameter was lowered from a mean value of 22.7 +/- 0.3% in susceptible sheep to 20.0 +/- 0.3% in resistant sheep. A causal relationship between high fibre diameter variation and fleece-rot susceptibility is suggested. Sheep with irregular fibre size may retain free moisture in the fleece for longer, and thereby become more susceptible to fleece-rot than sheep with uniform fibre diameter, other predisposing factors being equal.

The significance of certain skin characters of sheep in resistance and susceptibility to fleece-rot and body strike.

Micro-anatomical differences in skin structure associated with resistance and susceptibility of sheep to fleece-rot and body strike were identified, and found to be of similar magnitude in 2 genetically divergent flocks of medium-woolled Merino ewes. Susceptible sheep were characterised by smaller follicle groups, resulting in higher densities of follicle populations with greater concentrations of the primary follicles (and sudoriferous glands) than in the resistant sheep. From these smaller follicle groups of susceptible sheep, thicker wool fibres grew than were found in the resistant sheep. The significance of these findings is discussed in relation to the pathogenesis of fleece-rot and body strike of sheep, and the value of the measured skin characters as a method for identifying resistant sheep.