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.

Mitochondrial genomic investigation reveals a clear association between species and genotypes of Lucilia and geographic origin in Australia.

BACKGROUND: Lucilia cuprina and L. sericata (family Calliphoridae) are globally significant ectoparasites of sheep. Current literature suggests that only one of these blowfly subspecies, L. cuprina dorsalis, is a primary parasite causing myiasis (flystrike) in sheep in Australia. These species and subspecies are difficult to distinguish using morphological features. Hence, being able to accurately identify blowflies is critical for diagnosis and for understanding their relationships with their hosts and environment. METHODS: In this study, adult blowflies (5 pools of 17 flies; n = 85) were collected from five locations in different states [New South Wales (NSW), Queensland (QLD), Tasmania (TAS), Victoria (VIC) and Western Australia (WA)] of Australia and their mitochondrial (mt) genomes were assembled. RESULTS: Each mt genome assembled was ~ 15 kb in size and encoded 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a control region. The Lucilia species mt genomes were conserved in structure, and the genes retained the same order and direction. The overall nucleotide composition was heavily biased towards As and Ts-77.7% of the whole genomes. Pairwise nucleotide diversity suggested divergence between Lucilia cuprina cuprina, L. c. dorsalis and L. sericata. Comparative analyses of these mt genomes with published data demonstrated that the blowflies collected from sheep farm in TAS clustered within a clade with L. sericata. The flies collected from an urban location in QLD were more closely related to L. sericata and represented the subspecies L. c. cuprina, whereas the flies collected from sheep farms in NSW, VIC and WA represented the subspecies L. c. dorsalis. CONCLUSIONS: Phylogenetic analyses of the mt genomes representing Lucilia from the five geographic locations in Australia supported the previously demonstrated paraphyly of L. cuprina with respect to L. sericata and revealed that L. c. cuprina is distinct from L. c. dorsalis and that L. c. cuprina is more closely related to L. sericata than L. c. dorsalis. The mt genomes reported here provide an important molecular resource to develop tools for species- and subspecies-level identification of Lucilia from different geographical regions across Australia.

Molecular Differentiation of Four Species of Oropsylla (Siphonaptera: Ceratophyllidae) Using PCR-Based Single Strand Conformation Polymorphism Analyses and DNA Sequencing.

It is often difficult to distinguish morphologically between closely related species of fleas (Siphonaptera). Morphological identification of fleas often requires microscopic examination of internal structures in specimens cleared using caustic solutions. This process degrades DNA and/or inhibits DNA extraction from specimens, which limits molecular-based studies on individual fleas and their microbiomes. Our objective was to distinguish between Oropsylla rupestris (Jordan), Oropsylla tuberculata (Baker), Oropsylla bruneri (Baker), and Oropsylla labis (Jordan & Rothschild) (Ceratophyllidae) using PCR-based single strand conformation polymorphism (SSCP) analyses and DNA sequencing. A 446 bp region of the nuclear 28S ribosomal RNA (rRNA) gene was used as the genetic marker. The results obtained for 36 reference specimens (i.e., fleas that were morphologically identified to species) revealed no intraspecific variation in DNA sequence, whereas the DNA sequences of the four species of Oropsylla differed from one another at two to six nucleotide positions. Each flea species also had a unique SSCP banding pattern. SSCP analyses were then used to identify another 84 fleas that had not been identified morphologically. DNA sequencing data confirmed the species identity of fleas subjected to SSCP. This demonstrates that PCR-SSCP combined with DNA sequencing of the 28S rRNA gene is a very effective approach for the delineation of four closely related species of flea.

Parasitology Education Before and After the COVID-19 Pandemic.

The COVID-19 pandemic has disrupted parasitology curricula worldwide, which is expected to lead to the reshaping of parasitology education. Here, we share our experiences of remote teaching and learning of veterinary parasitology and discuss opportunities offered by remote teaching during COVID-19 lockdowns, enabling the development of interactive online parasitology courses.

Major SCP/TAPS protein expansion in Lucilia cuprina is associated with novel tandem array organisation and domain architecture.

BACKGROUND: Larvae of the Australian sheep blowfly, Lucilia cuprina, parasitise sheep by feeding on skin excretions, dermal tissue and blood, causing severe damage known as flystrike or myiasis. Recent advances in -omic technologies and bioinformatic data analyses have led to a greater understanding of blowfly biology and should allow the identification of protein families involved in host-parasite interactions and disease. Current literature suggests that proteins of the SCP (Sperm-Coating Protein)/TAPS (Tpx-1/Ag5/PR-1/Sc7) (SCP/TAPS) superfamily play key roles in immune modulation, cross-talk between parasite and host as well as developmental and reproductive processes in parasites. METHODS: Here, we employed a bioinformatics workflow to curate the SCP/TAPS protein gene family in L. cuprina. Protein sequence, the presence and number of conserved CAP-domains and phylogeny were used to group identified SCP/TAPS proteins; these were compared to those found in Drosophila melanogaster to make functional predictions. In addition, transcription levels of SCP/TAPS protein-encoding genes were explored in different developmental stages. RESULTS: A total of 27 genes were identified as belonging to the SCP/TAPS gene family: encoding 26 single-domain proteins each with a single CAP domain and a solitary double-domain protein containing two conserved cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domains. Surprisingly, 16 SCP/TAPS predicted proteins formed an extended tandem array spanning a 53 kb region of one genomic region, which was confirmed by MinION long-read sequencing. RNA-seq data indicated that these 16 genes are highly transcribed in all developmental stages (excluding the embryo). CONCLUSIONS: Future work should assess the potential of selected SCP/TAPS proteins as novel targets for the control of L. cuprina and related parasitic flies of major socioeconomic importance.

Gene content evolution in the arthropods.

BACKGROUND: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. RESULTS: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. CONCLUSIONS: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

Identification of bacteria in the Rocky Mountain wood tick, Dermacentor andersoni, using single-strand conformation polymorphism (SSCP) and DNA sequencing.

PCR-based single-strand conformation polymorphism (SSCP) analyses combined with DNA sequencing of the prokaryotic 16S ribosomal (r) RNA gene encompassing the hypervariable V4 region was used to determine the bacterial composition of Rocky Mountain wood ticks (Dermacentor andersoni) attached to Richardson's ground squirrels (Urocitellus richardsonii) and questing on vegetation in southern Saskatchewan, Canada. The bacteria present in questing adult ticks from Saskatchewan Landing Provincial Park included Rickettsia peacockii, a Francisella-like endosymbiont (FLE) and an Arsenophonus-like endosymbiont. Bacteria in the adult and nymphal ticks attached to U. richardsonii collected from Beechy included R. peacockii, a FLE, and several other genera (e.g., Ralstonia, Sphingobium, Comamonas and Pseudomonas). The bacteria detected in D. andersoni in the present study are consistent with the findings of other studies that have characterized the microbiome of this tick species in the USA using next generation sequencing. This result demonstrates that the SSCP-based approach used in this study is cost- and time-effective for examining bacterial composition in ticks.

The Battle Against Flystrike - Past Research and New Prospects Through Genomics.

Flystrike, or cutaneous myiasis, is caused by blow fly larvae of the genus Lucilia. This disease is a major problem in countries with large sheep populations. In Australia, Lucilia cuprina (Wiedemann, 1830) is the principal fly involved in flystrike. While much research has been conducted on L. cuprina, including physical, chemical, immunological, genetic and biological investigations, the molecular biology of this fly is still poorly understood. The recent sequencing, assembly and annotation of the draft genome and analyses of selected transcriptomes of L. cuprina have given a first global glimpse of its molecular biology and insights into host-fly interactions, insecticide resistance genes and intervention targets. The present article introduces L. cuprina, flystrike and associated issues, details past control efforts and research foci, reviews salient aspects of the L. cuprina genome project and discusses how the new genomic and transcriptomic resources for this fly might accelerate fundamental molecular research of L. cuprina towards developing new methods for the treatment and control of flystrike.

A blow to the fly - Lucilia cuprina draft genome and transcriptome to support advances in biology and biotechnology.

The blow fly, Lucilia cuprina (Wiedemann, 1830) is a parasitic insect of major global economic importance. Maggots of this fly parasitize the skin of animal hosts, feed on excretions and tissues, and cause severe disease (flystrike or myiasis). Although there has been considerable research on L. cuprina over the years, little is understood about the molecular biology, biochemistry and genetics of this parasitic fly, as well as its relationship with its hosts and the disease that it causes. This situation might change with the recent report of the draft genome and transcriptome of this blow fly, which has given new and global insights into its biology, interactions with the host animal and aspects of insecticide resistance at the molecular level. This genomic resource will likely enable many fundamental and applied research areas in the future. The present article gives a background on L. cuprina and myiasis, a brief account of past and current treatment, prevention and control approaches, and provides a perspective on the impact that the L. cuprina genome should have on future research of this and related parasitic flies, and the design of new and improved interventions for myiasis.

Lucilia cuprina genome unlocks parasitic fly biology to underpin future interventions.

Lucilia cuprina is a parasitic fly of major economic importance worldwide. Larvae of this fly invade their animal host, feed on tissues and excretions and progressively cause severe skin disease (myiasis). Here we report the sequence and annotation of the 458-megabase draft genome of Lucilia cuprina. Analyses of this genome and the 14,544 predicted protein-encoding genes provide unique insights into the fly's molecular biology, interactions with the host animal and insecticide resistance. These insights have broad implications for designing new methods for the prevention and control of myiasis.

Histone deacetylase enzymes as drug targets for the control of the sheep blowfly, Lucilia cuprina.

The Australian sheep blowfly, Lucilia cuprina, is an ecto-parasite that causes significant economic losses in the sheep industry. Emerging resistance to insecticides used to protect sheep from this parasite is driving the search for new drugs that act via different mechanisms. Inhibitors of histone deacetylases (HDACs), enzymes essential for regulating eukaryotic gene transcription, are prospective new insecticides based on their capacity to kill human parasites. The blowfly genome was found here to contain five HDAC genes corresponding to human HDACs 1, 3, 4, 6 and 11. The catalytic domains of blowfly HDACs 1 and 3 have high sequence identity with corresponding human and other Dipteran insect HDACs (Musca domestica and Drosophila melanogaster). On the other hand, HDACs 4, 6 and 11 from the blowfly and the other Dipteran species showed up to 53% difference in catalytic domain amino acids from corresponding human sequences, suggesting the possibility of developing HDAC inhibitors specific for insects as desired for a commercial insecticide. Differences in transcription patterns for different blowfly HDACs through the life cycle, and between the sexes of adult flies, suggest different functions in regulating gene transcription within this organism and possibly different vulnerabilities. Data that supports HDACs as possible new insecticide targets is the finding that trichostatin A and suberoylanilide hydroxamic acid retarded growth of early instar blowfly larvae in vitro, and reduced the pupation rate. Trichostatin A was 8-fold less potent than the commercial insecticide cyromazine in inhibiting larval growth. Our results support further development of inhibitors of blowfly HDACs with selectivity over human and other mammalian HDACs as a new class of prospective insecticides for sheep blowfly.

Comparison of the DNA sequences and secondary structure of the mitochondrial 16S rRNA gene of Ixodes kingi, Ixodes sculptus and Ixodes angustus.

The DNA sequences and predicted secondary structure of domains IV and V of the mitochondrial (mt) 16S rRNA gene were compared for three species of Ixodes: Ixodes kingi, Ixodes sculptus and Ixodes angustus. Each species had a unique set of DNA sequences for the 16S gene. Many of the differences in DNA sequence within and among species occurred in a "hypervariable" region of domain V, and either represented partial or full compensatory base pair changes that maintained the helices within the secondary structure, or nucleotide alterations at unpaired positions that had no effect on the secondary structure. The results of the phylogenetic analyses revealed that I. kingi, I. sculptus and I. angustus were placed in a clade with some other species of the subgenera Pholeoixodes and Ixodiopsis. In addition, individuals of I. sculptus from Saskatchewan (Canada) and Colorado (USA) did not form a monophyletic clade, suggesting the possible existence of cryptic species.

Discovery of novel Rickettsiella spp. in ixodid ticks from Western Canada.

The genomic DNA from four species of ixodid ticks in western Canada was tested for the presence of Rickettsiella by PCR analyses targeting the 16S rRNA gene. Eighty-eight percent of the Ixodes angustus (n = 270), 43% of the I. sculptus (n = 61), and 4% of the I. kingi (n = 93) individuals examined were PCR positive for Rickettsiella, whereas there was no evidence for the presence of Rickettsiella in Dermacentor andersoni (n = 45). Three different single-strand conformation polymorphism profiles of the 16S rRNA gene were detected among amplicons derived from Rickettsiella-positive ticks, each corresponding to a different sequence type. Furthermore, each sequence type was associated with a different tick species. Phylogenetic analyses of sequence data of the 16S rRNA gene and three other genes (rpsA, gidA, and sucB) revealed that all three sequence types were placed in a clade that contained species and pathotypes of the genus Rickettsiella. The bacterium in I. kingi represented the sister taxon to the Rickettsiella in I. sculptus, and both formed a clade with Rickettsiella grylli from crickets (Gryllus bimaculatus) and "R. ixodidis" from I. woodi. In contrast, the Rickettsiella in I. angustus was not a member of this clade but was placed external to the clade comprising the pathotypes of R. popilliae. The results indicate the existence of at least two new species of Rickettsiella: one in I. angustus and another in I. kingi and I. sculptus. However, the Rickettsiella strains in I. kingi and I. sculptus may also represent different species because each had unique sequences for all four genes.

Mutation scanning-based identification of larval and nymphal ticks (Acari: Ixodidae) from Richardson's ground squirrels (Spermophilus richardsonii).

The objective of this study was to identify the tick species parasitizing Richardson's ground squirrels (Spermophilus richardsonii) in southern Saskatchewan (Canada). Morphological examination of the adult ticks revealed the presence of three tick species, Ixodes sculptus, Ixodes kingi and Dermacentor andersoni. However, given the difficulties in identifying some of the larval and nymphal (immature) ticks using this approach, PCR-based single-strand conformation polymorphism (SSCP) and DNA sequence analyses of a portion of the mitochondrial (mt) 16S rRNA gene were used to determine their species identity. The results showed that each tick species had a unique set of SSCP profiles and DNA sequences using this mt marker. The species identity of larval and nymphal ticks was determined based on a comparison of these profiles and sequences with those of morphologically-identified adults. The detection of three tick species, which are known vectors of disease-causing agents, on the same host has important implications for understanding the ecology of vector-borne diseases, and provides an opportunity to examine fundamental questions regarding the structure and composition of the bacterial communities (i.e., both endosymbiotic and pathogenic species) in these ticks. This study shows the utility and benefits of using the present molecular method for the accurate identification of ticks at any stage of development.

A novel Rickettsia species detected in Vole Ticks (Ixodes angustus) from Western Canada.

The genomic DNA of ixodid ticks from western Canada was tested by PCR for the presence of Rickettsia. No rickettsiae were detected in Ixodes sculptus, whereas 18% of the I. angustus and 42% of the Dermacentor andersoni organisms examined were PCR positive for Rickettsia. The rickettsiae from each tick species were characterized genetically using multiple genes. Rickettsiae within the D. andersoni organisms had sequences at four genes that matched those of R. peacockii. In contrast, the Rickettsia present within the larvae, nymphs, and adults of I. angustus had novel DNA sequences at four of the genes characterized compared to the sequences available from GenBank for all recognized species of Rickettsia and all other putative species within the genus. Phylogenetic analyses of the sequence data revealed that the rickettsiae in I. angustus do not belong to the spotted fever, transitional, or typhus groups of rickettsiae but are most closely related to "Candidatus Rickettsia kingi" and belong to a clade that also includes R. canadensis, "Candidatus Rickettsia tarasevichiae," and "Candidatus Rickettsia monteiroi."

Detection of a novel Rickettsia (Alphaproteobacteria: Rickettsiales) in rotund ticks (Ixodes kingi) from Saskatchewan, Canada.

A novel Rickettsia was detected in the rotund tick, Ixodes kingi Bishopp, 1911, based on comparative DNA sequence analyses of 4 genes; the rickettsial-specific 17-kDa antigen gene, citrate synthase gene (gltA), the outer surface membrane protein A gene (ompA), and the 16S rRNA gene. The rickettsiae in I. kingi differed in nucleotide sequence from those of other Rickettsia species by 5.8-18.3% for the 17-kDa gene, 0.9-13.9% for gltA, 5.5-22.8% for ompA, and 0.9-1.6% for the 16S rRNA gene. Phylogenetic analyses of the sequence data revealed that this putative new species of Rickettsia, provisionally named Candidatus Rickettsia kingi, does not belong to the spotted fever group or typhus group of rickettsiae, but represents a sister taxon to R. canadensis and Candidatus Rickettsia tarasevichiae. This novel Rickettsia was found in 60 of the 87 (69%) ticks examined, which included all feeding life cycle stages of I. kingi. Although adult I. kingi occasionally parasitize dogs and humans, it remains to be determined if this Rickettsia is pathogenic to these host species.

Ticks (Acari: Ixodidae) on small mammals in Kootenay National Park, British Columbia, Canada.

Two hundred and ninety-one ticks (i.e., 185 larvae, 72 nymphs, and 34 adults) were removed from 153 small mammals comprising six species collected in Verdant Forest, Numa Forest, and Marble Canyon within Kootenay National Park, British Columbia, Canada. Morphological examination and molecular analyses (i.e., polymerase chain reaction-single-strand conformation polymorphism [PCR-SSCP] and DNA sequencing of the mitochondrial 16S rRNA gene) of the ticks revealed that most individuals were Ixodes angustus Neumann. All life cycle stages of I. angustus were found primarily on southern red-backed voles, Clethrionomys gapperi (Vigors). Two Dermacentor andersoni Stiles females were also found on these small mammals. The results of the molecular analyses also revealed that there were three 16S haplotypes of I. angustus and two 16S haplotypes of D. andersoni. A comparison of available sequence data suggests genetic divergence between I. angustus near the western and eastern limits of the species distributional range in North America. Additional studies are needed to determine whether there are genetic differences between I. angustus from North America, Japan, and Russia, and whether there is geographical variation in the ability of ticks to transmit pathogens to their mammalian hosts.

Ticks feeding on northern pocket gophers (Thomomys talpoides) in central Saskatchewan and the unexpected detection of Ixodes scapularis larvae.

Morphological examination of ticks feeding on northern pocket gophers, Thomomys talpoides, near Clavet (Saskatchewan, Canada) revealed the presence of two genera, Ixodes and Dermacentor. All adult ticks collected were identified as I. kingi. Single strand conformation polymorphism (SSCP) analyses and DNA sequencing of the mitochondrial 16S rRNA gene confirmed the species identity of most Ixodes immatures as I. kingi (two nymphs and 82 larvae), and the Dermacentor immatures as D. variabilis (one nymph and one larva) and D. andersoni (three larvae). Six Ixodes larvae feeding on three T. talpoides individuals were identified as four different 16S haplotypes of I. scapularis, which was unexpected because there are no known established populations of this species in Saskatchewan. However, flagging for questing ticks and further examination of the ticks feeding on T. talpoides in two subsequent years failed to detect the presence of I. scapularis near Clavet, suggesting that there is no established population of I. scapularis in this area. Nonetheless, since I. scapularis is a vector of pathogenic agents, passive and active surveillance needs to be conducted in Saskatchewan on an ongoing basis to determine if this tick species and its associated pathogens become established within the province.

An assessment of genetic differences among ixodid ticks in a locus within the nuclear large subunit ribosomal RNA gene.

We examined the usefulness of the D3 domain and flanking core regions (=D3(+)) of the nuclear large subunit (LSU) ribosomal DNA as a genetic marker for species-level identification and the inference of evolutionary relationships of ixodid ticks. Genetic variation was also examined in relation to the secondary structure of the LSU rDNA. The results revealed a lack of sequence difference in the D3(+) among species of Dermacentor and among some species of Ixodes, demonstrating that this gene region is not suitable as a species marker for all species of ixodid ticks. Of the 45 variable nucleotide positions in the sequence alignment of the D3(+), 23 did not alter the secondary structure of the LSU rDNA, because they occurred in unpaired positions, whereas 16 represented partial or full compensatory changes which maintained the secondary structure. Six deletions in the D3(+) sequence of all Ixodes species examined resulted in a shorter d4_1 helix compared with that of other tick species. The results of the phylogenetic analyses also showed that the D3(+) is of limited value in resolving evolutionary relationships among ixodid ticks. In addition, we also demonstrated that the D3(+) of ascomycete fungi could also be amplified along with, or instead of, the D3(+) of some tick species, depending upon the primers used in PCR. Nonetheless, the D3(+) of the fungal contaminants are readily distinguished from the D3(+) of ixodid ticks because of a shorter length and the absence of helix d4_1 in the secondary structure of the LSU rDNA.