Wolbachia endosymbiont of the horn fly Haematobia irritans irritans: a supergroup A strain with multiple horizontally acquired cytoplasmic incompatibility genes

The horn fly, Haematobia irritans irritans, is a hematophagous parasite of livestock distributed throughout Europe, Africa, Asia, and the Americas. Welfare losses on livestock due to horn fly infestation are estimated to cost between USD 1-2.5 billion annually in North America and Brazil. The endosymbiotic bacterium Wolbachia pipientis is a maternally inherited manipulator of reproductive biology in arthropods and naturally infects laboratory colonies of horn flies from Kerrville, USA and Alberta, Canada, but has also been identified in wild-caught samples from Canada, USA, Mexico and Hungary. Re-assembly of PacBio long-read and Illumina genomic DNA libraries from the Kerrville H. i. irritans genome project allowed for a complete and circularised 1.3 Mb Wolbachia genome (wIrr). Annotation of wIrr yielded 1249 coding genes, 34 tRNAs, three rRNAs, and five prophage regions. Comparative genomics and whole genome Bayesian evolutionary analysis of wIrr compared to published Wolbachia genomes suggests that wIrr is most closely related to and diverged from Wolbachia supergroup A strains known to infect Drosophila spp. Whole-genome synteny analyses between wIrr and closely related genomes indicates that wIrr has undergone significant genome rearrangements while maintaining high nucleotide identity. Comparative analysis of the cytoplasmic incompatibility (CI) genes of wIrr suggests two phylogenetically distinct CI loci and acquisition of another CifB homolog from phylogenetically distant supergroup A Wolbachia strains suggesting horizontal acquisition of these loci. The wIrr genome provides a resource for future examination of the impact Wolbachia may have in both biocontrol and potential insecticide resistance of horn flies

Molecular markers reveal diversity in composition of Megastigmus (Hymenoptera: Megastigmidae) from eucalypt galls

Since outbreaks of the invasive blue gum chalcids Leptocybe spp. began, the genus Megastigmus (Hymenoptera: Megastigmidae) has been increasingly studied as containing potential biocontrol agents against these pests. Megastigmus species have been collected and described from Australia, the presumed origin of Leptocybe spp., with M. zvimendeli and M. lawsoni reported as Leptocybe spp. parasitoids established outside of Australia. Parasitic Megastigmus have been reported to occur locally in the Neotropics, Afrotropic, Palearctic, and Indomalaya biogeographic realms, and in many cases described as new to science. However, molecular tools have not been used in studying parasitic Megastigmus, and difficulties in morphological taxonomy have compromised further understanding of eucalypt-associated Megastigmus as well as the Megastigmus-Leptocybe association. In this study, we used molecular markers to study the species composition and phylogeny of Megastigmus collected from eucalypt galls in Australia and from Leptocybe spp. galls from South Africa, Kenya, Israel, China, and Vietnam. We record thirteen discrete species and a species complex associated with eucalypt galls. A summary of morphological characters is provided to assist morphological delimitation of the studied group. A phylogeny based on 28S rDNA identified species groups of importance to Leptocybe spp. biocontrol agents from four clades with nine species. Relationships between Megastigmus from eucalypt galls and their phytophagous congeners were unresolved. Further molecular work is needed to clarify the identity of many species

Polychaetes (Perinereis helleri) reared in sand beds filtering nutrients from shrimp (Penaeus monodon) culture ponds can transiently carry IHHNV

A polychaete-assisted sand filter (PASF) system has been developed to help remove nutrients from aquaculture pond wastewater whilst also producing polychaetes that are highly prized as bait by recreational anglers and as a dietary supplement to improve the fecundity of shrimp broodstock. Whilst rearing polychaetes in PASF beds offers potential to reduce impacts of sourcing them from the wild, the use of wastewater from ponds rearing shrimp such as Penaeus monodon will present a biosecurity risk of viruses being transferred to, and potentially amplified in, the worms. To assess such risks for transmitting infectious hypodermal and haematopoietic necrosis virus (IHHNV), groups of 3 or 4 PASF beds seeded with sand worm (Perinereis helleri) juveniles were supplied with wastewater from ponds of P. monodon with either high-load or low-load IHHNV infections. TaqMan real-time qPCR identified low loads of IHHNV (≤878 IHHNV DNA copies 200 ng−1 TNA) in most worms from PASF beds supplied wastewater from the high-load pond. IHHNV was either not detected or detected at the qPCR test sensitivity limits in worms from beds supplied wastewater from the low-load pond. Purging harvested worms of their gut contents in clean filtered seawater for 2 days significantly reduced IHHNV loads. Reverting PASF beds to clean seawater for 8 weeks before harvest also significantly reduced worm loads of IHHNV. Daily additions of a commercial probiotic to the sand bed surface for 4 weeks prior to clean seawater application provided no discernible benefit to IHHNV clearance. While clearly demonstrated to be capable of carrying IHHNV, the remediation measures examined suggest potential to ameliorate the infection transmission risks of P. helleri reared in PASF beds supplied with shrimp pond wastewater as a nutrient source

Assessment of genetic structure among Australian east coast populations of snapper Chrysophrys auratus (Sparidae)

Snapper Chrysophrys auratus is a high-value food fish in Australia targeted by both commercial and recreational fisheries. Along the east coast of Australia, fisheries are managed under four state jurisdictions (Queensland, Qld; New South Wales, NSW; Victoria, Vic.; and Tasmania, Tas.), each applying different regulations, although it is thought that the fisheries target the same biological stock. An allozyme-based study in the mid-1990s identified a weak genetic disjunction north of Sydney (NSW) questioning the single-stock hypothesis. This study, focused on east-coast C. auratus, used nine microsatellite markers to assess the validity of the allozyme break and investigated whether genetic structure exists further south. Nine locations were sampled spanning four states and over 2000 km, including sites north and south of the proposed allozyme disjunction. Analyses confirmed the presence of two distinct biological stocks along the east coast, with a region of genetic overlap around Eden in southern NSW, ~400 km south of the allozyme disjunction. The findings indicate that C. auratus off Vic. and Tas. are distinct from those in Qld and NSW. For the purpose of stock assessment and management, the results indicate that Qld and NSW fisheries are targeting a single biological stock.

Development and Validation of Novel PCR Assays for the Diagnosis of Bovine Stephanofilariasis and Detection of Stephanofilaria sp. Nematodes in Vector Flies

Background: Stephanofilaria spp. nematodes are associated with cutaneous lesions in cattle and other livestock and mammalian wildlife species. In Australia, Haematobia irritans exigua, commonly known as buffalo fly (BF) transmits a well-described but presently unnamed species of Stephanofilaria, which has been speculatively implicated in the aetiology of BF lesions. The sensitivity of current techniques for detecting Stephanofilaria spp. in skin lesions and vector species is low, and there is no genomic sequence for any member of the genus Stephanofilaria currently available in sequence databases. Methods: To develop molecular assays for the detection of the Australian Stephanofilaria sp., skin biopsies were collected from freshly slaughtered cattle with typical lesions near the medial canthus. Adult nematodes and microfilariae were isolated from the biopsies using a saline recovery technique. The nematodes were morphologically identified as Stephanofilaria sp. by scanning electron microscopy. DNA was extracted and the internal transcribed spacer 2 (ITS2) region of rDNA, and the cytochrome c oxidase subunit 1 (cox1) region of mtDNA was amplified and sequenced. Stephanofilaria sp. specific polymerase chain reaction (PCR) and qPCR assays (SYBR Green® and TaqMan™) were developed and optimised from the novel ITS2 sequence obtained. The specificity of each assay was confirmed by testing against nematode species Onchocerca gibsoni and Dirofilaria immitis, as well as host (bovine) and BF DNA. Results: Scanning electron microscopy of the anterior and posterior ends of isolated nematodes confirmed Stephanofilaria sp. A phylogenetic analysis of the cox1 sequence demonstrated that this species is most closely related to Thelazia callipaeda, a parasitic nematode that is a common cause of thelaziasis (or eyeworm infestation) in humans, dogs, and cats. Both conventional and qPCR assays specifically amplified DNA from Stephanofilaria sp. Conventional PCR, TaqMan™, and SYBR Green® assays were shown to detect 1 ng, 1 pg, and 100 fg of Stephanofilaria DNA, respectively. Both qPCR assays detected DNA from single Stephanofilaria microfilaria. Conclusion: Molecular diagnostic assays developed in this study showed high specificity and sensitivity for Stephanofilaria sp. DNA. The availability of an accurate and sensitive PCR assay for Stephanofilaria will assist in determining its role in the pathogenesis of cattle skin lesions, as well as in understanding its epidemiological dynamics. This assay may also have application for use in epidemiological studies with other species of Stephanofilaria, most particularly closely related S. stilesi, but this will require confirmation

The complexity of Rhipicephalus (Boophilus) microplus genome characterised through detailed analysis of two BAC clones

<p>Abstract</p> <p>Background</p> <p><it>Rhipicephalus (Boophilus) microplus (Rmi) </it>a major cattle ectoparasite and tick borne disease vector, impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crustaceans. Model arthropod genomes such as <it>Drosophila </it>and <it>Anopheles </it>are too taxonomically distant for a reference in tick genomic sequence analysis. This study focuses on the <it>de-novo </it>assembly of two <it>R. microplus </it>BAC sequences from the understudied <it>R microplus </it>genome. Based on available <it>R. microplus </it>sequenced resources and comparative analysis, tick genomic structure and functional predictions identify complex gene structures and genomic targets expressed during tick-cattle interaction.</p> <p>Results</p> <p>In our BAC analyses we have assembled, using the correct positioning of BAC end sequences and transcript sequences, two challenging genomic regions. Cot DNA fractions compared to the BAC sequences confirmed a highly repetitive BAC sequence BM-012-E08 and a low repetitive BAC sequence BM-005-G14 which was gene rich and contained short interspersed elements (SINEs). Based directly on the BAC and Cot data comparisons, the genome wide frequency of the SINE Ruka element was estimated. Using a conservative approach to the assembly of the highly repetitive BM-012-E08, the sequence was de-convoluted into three repeat units, each unit containing an 18S, 5.8S and 28S ribosomal RNA (rRNA) encoding gene sequence (rDNA), related internal transcribed spacer and complex intergenic region.</p> <p>In the low repetitive BM-005-G14, a novel gene complex was found between to 2 genes on the same strand. Nested in the second intron of a large 9 Kb <it>papilin </it>gene was a <it>helicase </it>gene. This <it>helicase </it>overlapped in two exonic regions with the <it>papilin</it>. Both these genes were shown expressed in different tick life stage important in ectoparasite interaction with the host. Tick specific sequence differences were also determined for the <it>papilin </it>gene and the protein binding sites of the 18S subunit in a comparison to <it>Bos taurus</it>.</p> <p>Conclusion</p> <p>In the absence of a sequenced reference genome we have assembled two complex BAC sequences, characterised novel gene structure that was confirmed by gene expression and sequencing analyses. This is the first report to provide evidence for 2 eukaryotic genes with exon regions that overlap on the same strand, the first to describe <it>Rhipicephalinae papilin</it>, and the first to report the complete ribosomal DNA repeated unit sequence structure for ticks. The Cot data estimation of genome wide sequence frequency means this research will underpin future efforts for genome sequencing and assembly of the <it>R. microplus </it>genome.</p

Assessment of genetic structure among Australian east coast populations of snapper Chrysophrys auratus (Sparidae)

Snapper Chrysophrys auratus is a high-value food fish in Australia targeted by both commercial and recreational fisheries. Along the east coast of Australia, fisheries are managed under four state jurisdictions (Queensland, Qld; New South Wales, NSW; Victoria, Vic.; and Tasmania, Tas.), each applying different regulations, although it is thought that the fisheries target the same biological stock. An allozyme-based study in the mid-1990s identified a weak genetic disjunction north of Sydney (NSW) questioning the single-stock hypothesis. This study, focused on east-coast C. auratus, used nine microsatellite markers to assess the validity of the allozyme break and investigated whether genetic structure exists further south. Nine locations were sampled spanning four states and over 2000 km, including sites north and south of the proposed allozyme disjunction. Analyses confirmed the presence of two distinct biological stocks along the east coast, with a region of genetic overlap around Eden in southern NSW, ~400 km south of the allozyme disjunction. The findings indicate that C. auratus off Vic. and Tas. are distinct from those in Qld and NSW. For the purpose of stock assessment and management, the results indicate that Qld and NSW fisheries are targeting a single biological stock.

Phenome-wide association analysis of LDL-cholesterol lowering genetic variants in PCSK9

BACKGROUND: We characterised the phenotypic consequence of genetic variation at the PCSK9 locus and compared findings with recent trials of pharmacological inhibitors of PCSK9. METHODS: Published and individual participant level data (300,000+ participants) were combined to construct a weighted PCSK9 gene-centric score (GS). Seventeen randomized placebo controlled PCSK9 inhibitor trials were included, providing data on 79,578 participants. Results were scaled to a one mmol/L lower LDL-C concentration. RESULTS: The PCSK9 GS (comprising 4 SNPs) associations with plasma lipid and apolipoprotein levels were consistent in direction with treatment effects. The GS odds ratio (OR) for myocardial infarction (MI) was 0.53 (95% CI 0.42; 0.68), compared to a PCSK9 inhibitor effect of 0.90 (95% CI 0.86; 0.93). For ischemic stroke ORs were 0.84 (95% CI 0.57; 1.22) for the GS, compared to 0.85 (95% CI 0.78; 0.93) in the drug trials. ORs with type 2 diabetes mellitus (T2DM) were 1.29 (95% CI 1.11; 1.50) for the GS, as compared to 1.00 (95% CI 0.96; 1.04) for incident T2DM in PCSK9 inhibitor trials. No genetic associations were observed for cancer, heart failure, atrial fibrillation, chronic obstructive pulmonary disease, or Alzheimer's disease - outcomes for which large-scale trial data were unavailable. CONCLUSIONS: Genetic variation at the PCSK9 locus recapitulates the effects of therapeutic inhibition of PCSK9 on major blood lipid fractions and MI. While indicating an increased risk of T2DM, no other possible safety concerns were shown; although precision was moderate

Phenome-wide association analysis of LDL-cholesterol lowering genetic variants in PCSK9

Abstract: Background: We characterised the phenotypic consequence of genetic variation at the PCSK9 locus and compared findings with recent trials of pharmacological inhibitors of PCSK9. Methods: Published and individual participant level data (300,000+ participants) were combined to construct a weighted PCSK9 gene-centric score (GS). Seventeen randomized placebo controlled PCSK9 inhibitor trials were included, providing data on 79,578 participants. Results were scaled to a one mmol/L lower LDL-C concentration. Results: The PCSK9 GS (comprising 4 SNPs) associations with plasma lipid and apolipoprotein levels were consistent in direction with treatment effects. The GS odds ratio (OR) for myocardial infarction (MI) was 0.53 (95% CI 0.42; 0.68), compared to a PCSK9 inhibitor effect of 0.90 (95% CI 0.86; 0.93). For ischemic stroke ORs were 0.84 (95% CI 0.57; 1.22) for the GS, compared to 0.85 (95% CI 0.78; 0.93) in the drug trials. ORs with type 2 diabetes mellitus (T2DM) were 1.29 (95% CI 1.11; 1.50) for the GS, as compared to 1.00 (95% CI 0.96; 1.04) for incident T2DM in PCSK9 inhibitor trials. No genetic associations were observed for cancer, heart failure, atrial fibrillation, chronic obstructive pulmonary disease, or Alzheimer’s disease – outcomes for which large-scale trial data were unavailable. Conclusions: Genetic variation at the PCSK9 locus recapitulates the effects of therapeutic inhibition of PCSK9 on major blood lipid fractions and MI. While indicating an increased risk of T2DM, no other possible safety concerns were shown; although precision was moderate